Nualpun Sirinupong

Crystal Structure of Cardiac-specific Histone Methyltransferase SmyD1 Reveals Unusual Active Site Architecture

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.

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.

Crystal Structures of Histone and p53 Methyltransferase SmyD2 Reveal a Conformational Flexibility of the Autoinhibitory C-Terminal Domain

SmyD2 belongs to a new class of chromatin regulators that control gene expression in heart development and tumorigenesis. Besides methylation of histone H3 K4, SmyD2 can methylate non-histone targets including p53 and the retinoblastoma tumor suppressor. The methyltransferase activity of SmyD proteins has been proposed to be regulated by autoinhibition via the intra- and interdomain bending of the conserved C-terminal domain (CTD). However, there has been no direct evidence of a conformational change in the CTD. Here, we report two crystal structures of SmyD2 bound either to the cofactor product S-adenosylhomocysteine or to the inhibitor sinefungin. SmyD2 has a two-lobed structure with the active site located at the bottom of a deep crevice formed between the CTD and the catalytic domain. By extensive engagement with the methyltransferase domain, the CTD stabilizes the autoinhibited conformation of SmyD2 and restricts access to the catalytic site. Unexpectedly, despite that the two SmyD2 structures are highly superimposable, significant differences are observed in the first two helices of the CTDs: the two helices bend outwards and move away from the catalytic domain to generate a less closed conformation in the sinefungin-bound structure. Although the overall fold of the individual domains is structurally conserved among SmyD proteins, SmyD2 appear to be a conformational “intermediate” between a close form of SmyD3 and an open form of SmyD1. In addition, the structures reveal that the CTD is structurally similar to tetratricopeptide repeats (TPR), a motif through which many cochaperones bind to the heat shock protein Hsp90. Our results thus provide the first evidence for the intradomain flexibility of the TPR-like CTD, which may be important for the activation of SmyD proteins by Hsp90.

Structural insights into estrogen receptor α methylation by histone methyltransferase SMYD2, a cellular event implicated in estrogen signaling regulation

Estrogen receptor (ER) signaling plays a pivotal role in many developmental processes and has been implicated in numerous diseases including cancers. We recently showed that direct ERα methylation by the multi-specificity histone lysine methyltransferase SMYD2 regulates estrogen signaling through repressing ERα-dependent transactivation. However, the mechanism controlling the specificity of the SMYD2-ERα interaction and the structural basis of SMYD2 substrate binding diversity are unknown. Here we present the crystal structure of SMYD2 in complex with a target lysine (Lys266)-containing ERα peptide. The structure reveals that ERα binds SMYD2 in a U-shaped conformation with the binding specificity determined mainly by residues C-terminal to the target lysine. The structure also reveals numerous intrapeptide contacts that ensure shape complementarity between the substrate and the active site of the enzyme, thereby likely serving as an additional structural determinant of substrate specificity. In addition, comparison of the SMYD2-ERα and SMYD2-p53 structures provides the first structural insight into the diverse nature of SMYD2 substrate recognition and suggests that the broad specificity of SMYD2 is achieved by multiple molecular mechanisms such as distinct peptide binding modes and the intrinsic dynamics of peptide ligands. Strikingly, a novel potentially SMYD2-specific polyethylene glycol binding site is identified in the CTD domain, implicating possible functions in extended substrate binding or protein-protein interactions. Our study thus provides the structural basis for the SMYD2-mediated ERα methylation, and the resulting knowledge of SMYD2 substrate specificity and target binding diversity could have important implications in selective drug design against a wide range of ERα-related diseases.

Structural Insights into Neutrophilic Migration Revealed by the Crystal Structure of the Chemokine Receptor CXCR2 in Complex with the First PDZ Domain of NHERF1.

Neutrophil plays an essential role in host defense against infection, but uncontrolled neutrophilic infiltration can cause inflammation and severe epithelial damage. We recently showed that CXCR2 formed a signaling complex with NHERF1 and PLC-2, and that the formation of this complex was required for intracellular calcium mobilization and neutrophilic transepithelial migration. To uncover the structural basis of the complex formation, we report here the crystal structure of the NHERF1 PDZ1 domain in complex with the C-terminal sequence of CXCR2 at 1.16 Å resolution. The structure reveals that the CXCR2 peptide binds to PDZ1 in an extended conformation with the last four residues making specific side chain interactions. Remarkably, comparison of the structure to previously studied PDZ1 domains has allowed the identification of PDZ1 ligand-specific interactions and the mechanisms that govern PDZ1 target selection diversities. In addition, we show that CXCR2 can bind both NHERF1 PDZ1 and PDZ2 in pulldown experiments, consistent with the observation that the peptide binding pockets of these two PDZ domains are highly structurally conserved. The results of this study therefore provide structural basis for the CXCR2-mediated neutrophilic migration and could have important clinical applications in the prevention and treatment of numerous neutrophil-dependent inflammatory disorders.

Structural insights into PDZ-mediated interaction of NHERF2 and LPA(2), a cellular event implicated in CFTR channel regulation.

The formation of CFTR-NHERF2-LPA2 macromolecular complex in airway epithelia regulates CFTR channel function and plays an important role in compartmentalized cAMP signaling. We previously have shown that disruption of the PDZ-mediated NHERF2-LPA2 interaction abolishes the LPA inhibitory effect and augments CFTR Cl(-) channel activity in vitro and in vivo. Here we report the first crystal structure of the NHERF2 PDZ1 domain in complex with the C-terminal LPA2 sequence. The structure reveals that the PDZ1-LPA2 binding specificity is achieved by numerous hydrogen bonds and hydrophobic contacts with the last four LPA2 residues contributing to specific interactions. Comparison of the PDZ1-LPA2 structure to the structure of PDZ1 in complex with a different peptide provides insights into the diverse nature of PDZ1 substrate recognition and suggests that the conformational flexibility in the ligand binding pocket is involved in determining the broad substrate specificity of PDZ1. In addition, the structure reveals a small surface pocket adjacent to the ligand-binding site, which may have therapeutic implications. This study provides an understanding of the structural basis for the PDZ-mediated NHERF2-LPA2 interaction that could prove valuable in selective drug design against CFTR-related human diseases.

Crystallographic analysis of NHERF1–PLCβ3 interaction provides structural basis for CXCR2 signaling in pancreatic cancer

The formation of CXCR2-NHERF1-PLCβ3 macromolecular complex in pancreatic cancer cells regulates CXCR2 signaling activity and plays an important role in tumor proliferation and invasion. We previously have shown that disruption of the NHERF1-mediated CXCR2-PLCβ3 interaction abolishes the CXCR2 signaling cascade and inhibits pancreatic tumor growth in vitro and in vivo. Here we report the crystal structure of the NHERF1 PDZ1 domain in complex with the C-terminal PLCβ3 sequence. The structure reveals that the PDZ1-PLCβ3 binding specificity is achieved by numerous hydrogen bonds and hydrophobic contacts with the last four PLCβ3 residues contributing to specific interactions. We also show that PLCβ3 can bind both NHERF1 PDZ1 and PDZ2 in pancreatic cancer cells, consistent with the observation that the peptide binding pockets of these PDZ domains are highly structurally conserved. This study provides an understanding of the structural basis for the PDZ-mediated NHERF1-PLCβ3 interaction that could prove valuable in selective drug design against CXCR2-related cancers.

New Conformational State of NHERF1-CXCR2 Signaling Complex Captured by Crystal Lattice Trapping

NHERF1 is a PDZ adaptor protein that scaffolds the assembly of diverse signaling complexes and has been implicated in many cancers. However, little is known about the mechanism responsible for its scaffolding promiscuity or its ability to bind to multiple targets. Computational studies have indicated that PDZ promiscuity may be attributed to its conformational dynamics, but experimental evidence for this relationship remains very limited. Here we examine the conformational flexibility of the NHERF1 PDZ1 domain using crystal lattice trapping via solving PDZ1 structure of a new crystal form. The structure, together with prior PDZ1 structures of a different space group, reveals that 4 of 11 ligand-interacting residues undergo significant crystal packing-induced structural changes. Most of these residues correspond to the residues involved in allosteric transition when a peptide ligand binds. In addition, a subtle difference in ligand conformations causes the same peptide to bind in slightly different modes in different crystal forms. These findings indicate that substantial structural flexibility is present in the PDZ1 peptide-binding pocket, and the structural substate trapped in the present crystal form can be utilized to represent the conformational space accessible to the protein. Such knowledge will be critical for drug design against the NHERF1 PDZ1 domain, highlighting the continued need for experimentally determined PDZ1-ligand complexes.

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.

Crystal structure of the NHERF1 PDZ2 domain in complex with the chemokine receptor CXCR2 reveals probable modes of PDZ2 dimerization.

The formation of CXCR2-NHERF1-PLCβ2 macromolecular complex in neutrophils regulates CXCR2 signaling and plays a key role in neutrophil chemotaxis and transepithelial neutrophilic migration. However, NHERF1 by itself, with only two PDZ domains, has a limited capacity in scaffolding the multiprotein-complex formation. Here we report the crystal structure of the NHERF1 PDZ2 domain in complex with the C-terminal CXCR2 sequence. The structure reveals that the PDZ2-CXCR2 binding specificity is achieved by numerous hydrogen bonds and hydrophobic contacts with the last four CXCR2 residues contributing to specific interactions. The structure also reveals two probable modes of PDZ2 dimerization where the two canonical ligand-binding pockets are well separated and orientated in a unique parallel fashion. This study provides not only the structural basis for the PDZ-mediated NHERF1-CXCR2 interaction, but also an additional example of how PDZ domains may dimerize, which both could prove valuable in understanding NHERF1 complex-scaffolding function in neutrophils.