NAD(H) phosphates mediate tetramer assembly of human C-terminal binding protein (CtBP).

C-terminal binding proteins (CtBPs) are cotranscriptional factors that play key roles in cell fate. We have previously shown that NAD(H) promotes the assembly of similar tetramers from either human CtBP1 and CtBP2 and that CtBP2 tetramer destabilizing mutants are defective for oncogenic activity. To assist structure-based design efforts for compounds that disrupt CtBP tetramerization, it is essential to understand how NAD(H) triggers tetramer assembly.

Cryo-EM structure of CtBP2 confirms tetrameric architecture.

C-terminal binding proteins 1 and 2 (CtBP1 and CtBP2) are transcriptional regulators that activate or repress many genes involved in cellular development, apoptosis, and metastasis. NADH-dependent CtBP activation has been implicated in multiple types of cancer and poor patient prognosis. Central to understanding activation of CtBP in oncogenesis is uncovering how NADH triggers protein assembly, what level of assembly occurs, and if oncogenic activity depends upon such assembly.

Optimizing the refinement of merohedrally twinned P6 HIV-1 protease-inhibitor cocrystal structures.

Twinning is a crystal-growth anomaly in which protein monomers exist in different orientations but are related in a specific way, causing diffraction reflections to overlap. Twinning imposes additional symmetry on the data, often leading to the assignment of a higher symmetry space group. Specifically, in merohedral twinning, reflections from each monomer overlap and require a twin law to model unique structural data from overlapping reflections.

Active-Site Tryptophan, the Target of Antineoplastic C-Terminal Binding Protein Inhibitors, Mediates Inhibitor Disruption of CtBP Oligomerization and Transcription Coregulatory Activities.

C-terminal binding proteins (CtBP1/2) are oncogenic transcriptional coregulators and dehydrogenases often overexpressed in multiple solid tumors, including breast, colon, and ovarian cancer, and associated with poor survival. CtBPs act by repressing expression of genes responsible for apoptosis (e.g.

Assembly of human C-terminal binding protein (CtBP) into tetramers.

C-terminal binding protein 1 (CtBP1) and CtBP2 are transcriptional coregulators that repress numerous cellular processes, such as apoptosis, by binding transcription factors and recruiting chromatin-remodeling enzymes to gene promoters. The NAD(H)-linked oligomerization of human CtBP is coupled to its co-transcriptional activity, which is implicated in cancer progression. However, the biologically relevant level of CtBP assembly has not been firmly established; nor has the stereochemical arrangement of the subunits above that of a dimer.

Crystal structure of APOBEC3A bound to single-stranded DNA reveals structural basis for cytidine deamination and specificity.

Nucleic acid editing enzymes are essential components of the immune system that lethally mutate viral pathogens and somatically mutate immunoglobulins, and contribute to the diversification and lethality of cancers. Among these enzymes are the seven human APOBEC3 deoxycytidine deaminases, each with unique target sequence specificity and subcellular localization. While the enzymology and biological consequences have been extensively studied, the mechanism by which APOBEC3s recognize and edit DNA remains elusive.

Design, synthesis, and biological evaluation of substrate-competitive inhibitors of C-terminal Binding Protein (CtBP).

C-terminal Binding Protein (CtBP) is a transcriptional co-regulator that downregulates the expression of many tumor-suppressor genes. Utilizing a crystal structure of CtBP with its substrate 4-methylthio-2-oxobutyric acid (MTOB) and NAD(+) as a guide, we have designed, synthesized, and tested a series of small molecule inhibitors of CtBP. From our first round of compounds, we identified 2-(hydroxyimino)-3-phenylpropanoic acid as a potent CtBP inhibitor (IC50=0.

Structure-guided design of a high affinity inhibitor to human CtBP.

Oncogenic transcriptional coregulators C-terminal Binding Protein (CtBP) 1 and 2 possess regulatory d-isomer specific 2-hydroxyacid dehydrogenase (D2-HDH) domains that provide an attractive target for small molecule intervention. Findings that the CtBP substrate 4-methylthio 2-oxobutyric acid (MTOB) can interfere with CtBP oncogenic activity in cell culture and in mice confirm that such inhibitors could have therapeutic benefit. Recent crystal structures of CtBP 1 and 2 revealed that MTOB binds in an active site containing a dominant tryptophan and a hydrophilic cavity, neither of which are present in other D2-HDH family members.

Insight into the allosteric mechanism of Scapharca dimeric hemoglobin.

Allosteric regulation is an essential function of many proteins that control a variety of different processes such as catalysis, signal transduction, and gene regulation. Structural rearrangements have historically been considered the main means of communication between different parts of a protein. Recent studies have highlighted the importance, however, of changes in protein flexibility as an effective way to mediate allosteric communication across a protein.

Crystal structures of human CtBP in complex with substrate MTOB reveal active site features useful for inhibitor design.

The oncogenic corepressors C-terminal Binding Protein (CtBP) 1 and 2 harbor regulatory d-isomer specific 2-hydroxyacid dehydrogenase (d2-HDH) domains. 4-Methylthio 2-oxobutyric acid (MTOB) exhibits substrate inhibition and can interfere with CtBP oncogenic activity in cell culture and mice. Crystal structures of human CtBP1 and CtBP2 in complex with MTOB and NAD(+) revealed two key features: a conserved tryptophan that likely contributes to substrate specificity and a hydrophilic cavity that links MTOB with an NAD(+) phosphate.

Resolution of structural heterogeneity in dynamic crystallography.

Dynamic behavior of proteins is critical to their function. X-ray crystallography, a powerful yet mostly static technique, faces inherent challenges in acquiring dynamic information despite decades of effort. Dynamic `structural changes' are often indirectly inferred from `structural differences' by comparing related static structures.

Tertiary and quaternary allostery in tetrameric hemoglobin from Scapharca inaequivalvis.

The clam Scapharca inaequivalvis possesses two cooperative oxygen binding hemoglobins in its red cells: a homodimeric HbI and a heterotetrameric A2B2 HbII. Each AB dimeric half of HbII is assembled in a manner very similar to that of the well-studied HbI. This study presents crystal structures of HbII along with oxygen binding data both in the crystalline state and in wet nanoporous silica gels.

Cooperative macromolecular device revealed by meta-analysis of static and time-resolved structures.

Here we present a meta-analysis of a large collection of static structures of a protein in the Protein Data Bank in order to extract the progression of structural events during protein function. We apply this strategy to the homodimeric hemoglobin HbI from Scapharca inaequivalvis. We derive a simple dynamic model describing how binding of the first ligand in one of the two chemically identical subunits facilitates a second binding event in the other partner subunit.

Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding.

Hepatitis C virus infects an estimated 180 million people worldwide, prompting enormous efforts to develop inhibitors targeting the essential NS3/4A protease. Resistance against the most promising protease inhibitors, telaprevir, boceprevir, and ITMN-191, has emerged in clinical trials. In this study, crystal structures of the NS3/4A protease domain reveal that viral substrates bind to the protease active site in a conserved manner defining a consensus volume, or substrate envelope.

Crystal structure of the APOBEC3G catalytic domain reveals potential oligomerization interfaces.

APOBEC3G is a DNA cytidine deaminase that has antiviral activity against HIV-1 and other pathogenic viruses. In this study the crystal structure of the catalytically active C-terminal domain was determined to 2.25 A.

Structural insights into interferon regulatory factor activation.

The interferon regulatory factors (IRFs) play important roles in development of the immune system and host defense. Recent crystallographic and biochemical studies have provided insights into the mechanism of activation of IRFs by phosphorylation. The activation of a latent closed conformation of IRF in the cytoplasm is triggered by phosphorylation of Ser/Thr residues in a C-terminal region.

Ligand migration and cavities within Scapharca Dimeric HbI: studies by time-resolved crystallo-graphy, Xe binding, and computational analysis.

As in many other hemoglobins, no direct route for migration of ligands between solvent and active site is evident from crystal structures of Scapharca inaequivalvis dimeric HbI. Xenon (Xe) and organic halide binding experiments, along with computational analysis presented here, reveal protein cavities as potential ligand migration routes. Time-resolved crystallographic experiments show that photodissociated carbon monoxide (CO) docks within 5 ns at the distal pocket B site and at more remote Xe4 and Xe2 cavities.

Insights into interferon regulatory factor activation from the crystal structure of dimeric IRF5.

Interferon regulatory factors (IRFs) are essential in the innate immune response and other physiological processes. Activation of these proteins in the cytoplasm is triggered by phosphorylation of serine and threonine residues in a C-terminal autoinhibitory region, which stimulates dimerization, transport into the nucleus, assembly with the coactivator CBP/p300 and initiation of transcription. The crystal structure of the transactivation domain of pseudophosphorylated human IRF5 strikingly reveals a dimer in which the bulk of intersubunit interactions involve a highly extended C-terminal region.

Contribution of Ser386 and Ser396 to activation of interferon regulatory factor 3.

IRF-3, a member of the interferon regulatory factor (IRF) family of transcription factors, functions in innate immune defense against viral infection. Upon infection, host cell IRF-3 is activated by phosphorylation at its seven C-terminal Ser/Thr residues: (385)SSLENTVDLHISNSHPLSLTS(405). This phosphoactivation triggers IRF-3 to react with the coactivators, CREB-binding protein (CBP)/p300, to form a complex that activates target genes in the nucleus.

Time-resolved x-ray crystallography of heme proteins.

Heme proteins, with their natural photosensitivity, are excellent systems for the application of time-resolved crystallographic methods. Ligand dissociation can be readily initiated by a short laser pulse with global structural changes probed at the atomic level by X-rays in real time. Third-generation synchrotrons provide 100-ps X-ray pulses of sufficient intensity for monitoring very fast processes.

Ligand migration and binding in the dimeric hemoglobin of Scapharca inaequivalvis.

Using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) at cryogenic temperatures, we have studied CO binding to the heme and CO migration among cavities in the interior of the dimeric hemoglobin of Scapharca inaequivalvis (HbI) after photodissociation. By combining these studies with X-ray crystallography, three transient ligand docking sites were identified: a primary docking site B in close vicinity to the heme iron, and two secondary docking sites C and D corresponding to the Xe4 and Xe2 cavities of myoglobin. To assess the relevance of these findings for physiological binding, we also performed flash photolysis experiments on HbICO at room temperature and equilibrium binding studies with dioxygen.

Competition between Ski and CREB-binding protein for binding to Smad proteins in transforming growth factor-beta signaling.

The family of Smad proteins mediates transforming growth factor-beta (TGF-beta) signaling in cell growth and differentiation. Smads repress or activate TGF-beta signaling by interacting with corepressors (e.g.

An optical signal correlated with the allosteric transition in Scapharca inaequivalvis HbI.

The transient absorbance change within the first 2 mus of photolysis of COHbI (from Scapharca inaequivalvis) reported by Chiancone et al. [Chiancone, E., Elber, R.