Biomolecular dynamics in the 21st century.

The relevance of motions in biological macromolecules has been clear since the early structural analyses of proteins by X-ray crystallography. Computer simulations have been applied to provide a deeper understanding of the dynamics of biological macromolecules since 1976, and are now a standard tool in many labs working on the structure and function of biomolecules. In this mini-review we highlight some areas of current interest and active development for simulations, in particular all-atom molecular dynamics simulations.

Syk inhibits the activity of protein kinase A by phosphorylating tyrosine 330 of the catalytic subunit.

The Syk protein-tyrosine kinase can have multiple effects on cancer cells, acting in some as a tumor suppressor by inhibiting motility and in others as a tumor promoter by enhancing survival. Phosphoproteomic analyses identified PKA as a Syk-specific substrate. Syk catalyzes the phosphorylation of the catalytic subunit of PKA (PKAc) both in vitro and in cells on Tyr-330. Tyr-330 lies within the adenosine-binding motif in the C-terminal tail of PKAc within a cluster of acidic amino acids (DDYEEEE), which is a characteristic of Syk substrates. The phosphorylation of PKAc on Tyr-330 by Syk strongly inhibits its catalytic activity. Molecular dynamics simulations suggest that this additional negative charge prevents the C-terminal tail from interacting with the substrate and the nucleotide-binding site to stabilize the closed conformation of PKAc, thus preventing catalysis from occurring. Phosphoproteomic analyses and Western blotting studies indicate that Tyr-330 can be phosphorylated in a Syk-dependent manner in MCF7 breast cancer cells and DT40 B cells. The phosphorylation of a downstream substrate of PKAc, cAMP-responsive element-binding protein (CREB), is inhibited in cells expressing Syk but can be rescued by a selective inhibitor of Syk. Modulation of CREB activity alters the expression of the CREB-regulated gene BCL2 and modulates cellular responses to genotoxic agents. Thus, PKA is a novel substrate of Syk, and its phosphorylation on Tyr-330 inhibits its participation in downstream signaling pathways.

Identification of a novel effector domain of BIN1 for cancer suppression.

Bridging integrator 1 (BIN1) is a nucleocytoplasmic adaptor protein with tumor suppressor properties. The protein interacts with and inhibits the c-MYC transcription factor through the BIN1 MYC-binding domain (MBD). However, in vitro colony formation assays have clearly demonstrated that the MBD is not essential for BIN1-mediated growth arrest. We hypothesized that BIN1 contains a MYC-independent effector domain (MID) for cancer suppression. Because a functionally unique domain frequently contains a distinct structure, the human full-length BIN1 protein was subjected to limited trypsin digestion and the digested peptides were analyzed with Edman sequencing and mass spectrometry. We identified a trypsin-resistant peptide that corresponds to amino acids 146-268 of BIN1. It encompassed part of the BAR region, a putative effector region of BIN1. Computational analysis predicted that the peptide is very likely to exhibit coiled-coil motifs, implying a potential role for this region in sustaining the BIN1 structure and function. Like MBD-deleted BIN1, the trypsin-resistant peptide of BIN1 was predominantly present in the cytoplasm and was sufficient to inhibit cancer growth, regardless of dysregulated c-MYC activity. Our results suggest that the coiled-coil BIN1 BAR peptide encodes a novel BIN1 MID domain, through which BIN1 acts as a MYC-independent cancer suppressor.

Design, synthesis, and preclinical evaluation of prostate-specific membrane antigen targeted (99m)Tc-radioimaging agents.

The high mortality and financial burden associated with prostate cancer can be partly attributed to a lack of sensitive screening methods for detection and staging of the disease. Guided by in silico docking studies using the crystal structure of PSMA, we designed and synthesized a series of PSMA-targeted (99m)Tc-chelate complexes for imaging PSMA-expressing human prostate cancer cells (LNCaP cell line). Of the six targeted radioimaging agents synthesized, three were found to bind LNCaP cells with low nanomolar affinity. Moreover, the same three PSMA-targeted imaging agents were shown to localize primarily to LNCaP tumor xenografts in nu/nu mice, with an average of 9.8 +/- 2.4% injected dose/g tissue accumulating in the tumor and only 0.11% injected dose/g tissue retained in the muscle at 4 h postinjection. Collectively, these high affinity, PSMA-specific radioimaging agents demonstrate significant potential for use in localizing prostate cancer masses, monitoring response to therapy, detecting prostate cancer recurrence following surgery, and selecting patients for subsequent PSMA-targeted chemotherapy.

Molecular basis for a direct interaction between the Syk protein-tyrosine kinase and phosphoinositide 3-kinase.

After engagement of the B cell receptor for antigen, the Syk protein-tyrosine kinase becomes phosphorylated on multiple tyrosines, some of which serve as docking sites for downstream effectors with SH2 or other phosphotyrosine binding domains. The most frequently identified binding partner for catalytically active Syk identified in a yeast two-hybrid screen was the p85 regulatory subunit of phosphoinositide 3-kinase. The C-terminal SH2 domain of p85 was sufficient for mediating an interaction with tyrosine-phosphorylated Syk. Interestingly, this domain interacted with Syk at phosphotyrosine 317, a site phosphorylated in trans by the Src family kinase, Lyn, and identified previously as a binding site for c-Cbl. This site interacted preferentially with the p85 C-terminal SH2 domain compared with the c-Cbl tyrosine kinase binding domain. Molecular modeling studies showed a good fit between the p85 SH2 domain and a peptide containing phosphotyrosine 317. Tyr-317 was found to be essential for Syk to support phagocytosis mediated by FcgammaRIIA receptors expressed in a heterologous system. These studies establish a new type of p85 binding site that can exist on proteins that serve as substrates for Src family kinases and provide a molecular explanation for observations on direct interactions between Syk and phosphoinositide 3-kinase.

Flavivirus capsid is a dimeric alpha-helical protein.

The capsid proteins of two flaviviruses, yellow fever virus and dengue virus, were expressed in Escherichia coli and purified to near homogeneity suitable for biochemical characterization and structure determination by nuclear magnetic resonance. The oligomeric properties of the capsid protein in solution were investigated. In the absence of nucleic acid, both proteins were predominantly dimeric in solution. Further analysis of both proteins with far-UV circular dichroism spectroscopy indicated that they were largely alpha-helical. The secondary structure elements of the dengue virus capsid were determined by chemical shift indexing of the sequence-specific backbone resonance assignments. The dengue virus capsid protein devoid of its C-terminal signal sequence was found to be composed of four alpha helices. The longest alpha helix, 20 residues, is located at the C terminus and has an amphipathic character. In contrast, the N terminus was found to be unstructured and could be removed without disrupting the structural integrity of the protein.