The 2.4-A crystal structure of Scapharca dimeric hemoglobin. Cooperativity based on directly communicating hemes at a novel subunit interface.

The crystal structure of the cooperative dimeric hemoglobin from the arcid clam, Scapharca inaequivalvis, has been determined in the carbonmonoxy state. The phase problem was solved for reflections with Bragg spacings greater than 3 A using anomalous scattering from the porphyrin iron atoms measured at a single wavelength in combination with molecular averaging. The model built into this electron density map has been refined at 2.4 A resolution by means of stereochemically restrained least squares minimization to a conventional R-value of 0.156. The root mean square deviation from ideal bond lengths and angles are 0.013 A and 1.7 °, respectively. In addition to the 2336 hemoglobin atoms, 214 water molecules have been incorporated into the model. This structure reveals the details of an assemblage of two identical myoglobin-like subunits that is radically different from vertebrate hemoglobins. The subunit interface is formed by direct apposition of the E and F helices, whereas these surfaces are external in vertebrate hemoglobins. The interface has both hydrophobic and hydrophilic character. Two symmetrically related hydrophobic regions are formed between subunits. Six residues are involved in each of these regions that pack tightly enough to exclude water but have only a few atoms in close van der Waals contact. A number of ordered water molecules line the interface and form bridging hydrogen bonds between subunits. Four intersubunit ionic interactions are formed, two of which involve negatively charged propionate groups of the porphyrin. In contrast to cooperative vertebrate hemoglobins, a hydrogen bond network provides a direct route for communication between the two heme groups

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

C-terminal binding proteins (CtBPs) are co-transcriptional 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. Here, we investigate the moieties within NAD(H) that are responsible for triggering tetramer formation. Using multi-angle light scattering (MALS) we show that ADP is able to promote tetramer formation of both CtBP1 and CtBP2, whereas AMP promotes tetramer assembly of CtBP1, but not CtBP2. Other NAD(H) moieties that lack the adenosine phosphate, including adenosine and those incorporating nicotinamide, all fail to promote tetramer assembly. Our crystal structures of CtBP1 with AMP reveal participation of the adenosine phosphate in the tetrameric interface, pinpointing its central role in NAD(H) linked assembly. CtBP1 and CtBP2 have overlapping but unique roles, suggesting that a detailed understanding of their unique structural properties might have utility in the design of paralog specific inhibitors. We investigated the different responses to AMP through a series of site-directed mutants at 13 positions. These mutations reveal a central role for a hinge segment, which we term the 120s hinge, that connects the substrate with coenzyme binding domains and influences nucleotide binding and tetramer assembly. Our results provide insight into suitable pockets to explore in structure-based drug design to interfere with co-transcriptional activity of CtBP in cancer

Ligand binding and conformation change in the dimeric hemoglobin of the clam Scapharca inaequivalvis.

The reaction with carbon monoxide of the cooperative dimeric hemoglobin from Scapharca inaequivalvis has been examined by flash photolysis. In the nanosecond time range, geminate rebinding of 5% of dissociated CO occurs with a rate constant of 1.4 x 10(7) s-1. There is a change in absorbance of deoxyhemoglobin following photolysis at a rate of 1.2 x 10(6) s-1, consistent with a shift in the position of the Soret band to longer wavelengths. The amplitude of the change is proportional to the population of deoxydimer. In much of the Soret region this change is greater than the absorbance excursion associated with geminate recombination. There is at least one other slower change associated with the singly liganded species. Geminate rebinding of NO has components of 50, 8, and 0.035 ns-1, accounting for 75%, 25%, and less than 1% of the total reaction observed after a 35-ps photolysis flash. Simulation of diffusion of NO by molecular dynamics shows the ligands moving from the heme pocket to a subsidiary space between the edge of the heme and the surface of the protein

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. Here, multi-angle light scattering (MALS) data established the NAD(+)- and NADH-dependent assembly of CtBP1 and CtBP2 into tetramers. An examination of subunit interactions within CtBP1 and CtBP2 crystal lattices revealed that both share a very similar tetrameric arrangement resulting from assembly of two dimeric pairs, with specific interactions probably being sensitive to NAD(H) binding. Creating a series of mutants of both CtBP1 and CtBP2, we tested the hypothesis that the crystallographically observed interdimer pairing stabilizes the solution tetramer. MALS data confirmed that these mutants disrupt both CtBP1 and CtBP2 tetramers, with the dimer generally remaining intact, providing the first stereochemical models for tetrameric assemblies of CtBP1 and CtBP2. The crystal structure of a subtle destabilizing mutant suggested that small structural perturbations of the hinge region linking the substrate- and NAD-binding domains are sufficient to weaken the CtBP1 tetramer. These results strongly suggest that the tetramer is important in CtBP function, and the series of CtBP mutants reported here can be used to investigate the physiological role of the tetramer

Ligand Migration and Cavities within Scapharca Dimeric HbI: Studies by Time-Resolved Crystallo- graphy, Xe Binding, and Computational Analysis

SummaryAs 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. CO rebinding is not affected by the presence of dichloroethane within the major Xe4 protein cavity, demonstrating that this cavity is not on the major exit pathway. The crystal lattice has a substantial influence on ligand migration, suggesting that significant conformational rearrangements may be required for ligand exit. Taken together, these results are consistent with a distal histidine gate as one important ligand entry and exit route, despite its participation in the dimeric interface

CryoEM Structure of CtBP2 Confirms Tetrameric Architecture [preprint]

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. CtBP proteins are activated under hypoxic conditions where NAD(H) levels tend to be higher. NADH-dependent activation of CtBP2 has direct implication in multiple types of cancers and poor patient prognosis. Previous studies have proposed dimeric CtBP as the relevant oligomeric state, however our studies with multi-angle light scattering have shown that the primary effect of NADH binding is to promote the assembly of two CtBP dimers into tetramers. Here, we present the cryoEM structures of two different constructs of CtBP2 corroborating that the native state of CtBP2 in the presence of NADH is indeed tetrameric. The physiological relevance of tetrameric CtBP2 was tested in HCT116; CtBP2 -/- cells transfected with tetramer destabilizing mutants. Mutants that inhibit tetramer formation show a decrease in expression of the CtBP transcriptional target TIAM1 and exhibit a decrease in the ability to promote cell migration. Together with our cryoEM studies, these results highlight the tetramer as the functional oligomeric form of CtBP2

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. Here we present the crystal structure of a complex of a cytidine deaminase with ssDNA bound in the active site at 2.2 A. This structure not only visualizes the active site poised for catalysis of APOBEC3A, but pinpoints the residues that confer specificity towards CC/TC motifs. The APOBEC3A-ssDNA complex defines the 5\u27-3\u27 directionality and subtle conformational changes that clench the ssDNA within the binding groove, revealing the architecture and mechanism of ssDNA recognition that is likely conserved among all polynucleotide deaminases, thereby opening the door for the design of mechanistic-based therapeutics

Seasonal evolution of Aleutian low pressure systems: Implications for the North Pacific subpolar circulation

The seasonal change in the development of Aleutian low pressure systems from early fall to early winter is analyzed using a combination of meteorological reanalysis fields, satellite sea surface temperature (SST) data, and satellite wind data. The time period of the study is September–December 2002, although results are shown to be representative of the long-term climatology. Characteristics of the storms were documented as they progressed across the North Pacific, including their path, central pressure, deepening rate, and speed of translation. Clear patterns emerged. Storms tended to deepen in two distinct geographical locations—the Gulf of Alaska in early fall and the western North Pacific in late fall. In the Gulf of Alaska, a quasi-permanent “notch” in the SST distribution is argued to be of significance. The signature of the notch is imprinted in the atmosphere, resulting in a region of enhanced cyclonic potential vorticity in the lower troposphere that is conducive for storm development. Later in the season, as winter approaches and the Sea of Okhotsk becomes partially ice covered and cold, the air emanating from the Asian continent leads to enhanced baroclinicity in the region south of Kamchatka. This corresponds to enhanced storm cyclogenesis in that region. Consequently, there is a seasonal westward migration of the dominant lobe of the Aleutian low. The impact of the wind stress curl pattern resulting from these two regions of storm development on the oceanic circulation is investigated using historical hydrography. It is argued that the seasonal bimodal input of cyclonic vorticity from the wind may be partly responsible for the two distinct North Pacific subarctic gyres

The Wide-field Infrared Survey Explorer (WISE): Mission Description and Initial On-orbit Performance

The all sky surveys done by the Palomar Observatory Schmidt, the European Southern Observatory Schmidt, and the United Kingdom Schmidt, the InfraRed Astronomical Satellite and the 2 Micron All Sky Survey have proven to be extremely useful tools for astronomy with value that lasts for decades. The Wide-field Infrared Survey Explorer is mapping the whole sky following its launch on 14 December 2009. WISE began surveying the sky on 14 Jan 2010 and completed its first full coverage of the sky on July 17. The survey will continue to cover the sky a second time until the cryogen is exhausted (anticipated in November 2010). WISE is achieving 5 sigma point source sensitivities better than 0.08, 0.11, 1 and 6 mJy in unconfused regions on the ecliptic in bands centered at wavelengths of 3.4, 4.6, 12 and 22 microns. Sensitivity improves toward the ecliptic poles due to denser coverage and lower zodiacal background. The angular resolution is 6.1, 6.4, 6.5 and 12.0 arc-seconds at 3.4, 4.6, 12 and 22 microns, and the astrometric precision for high SNR sources is better than 0.15 arc-seconds.Comment: 22 pages with 19 included figures. Updated to better match the accepted version in the A

The Wide-Field Infrared Survey Explorer (WISE): Mission Description and Initial On-Orbit Performance

The all sky surveys done by the Palomar Observatory Schmidt, the European Southern Observatory Schmidt, and the United Kingdom Schmidt, the InfraRed Astronomical Satellite and the 2 Micron All Sky Survey have proven to be extremely useful tools for astronomy with value that lasts for decades. The Wide-field Infrared Survey Explorer is mapping the whole sky following its launch on 14 December 2009. WISE began surveying the sky on 14 Jan 2010 and completed its first full coverage of the sky on July 17. The survey will continue to cover the sky a second time until the cryogen is exhausted (anticipated in November 2010). WISE is achieving 5 sigma point source sensitivities better than 0.08, 0.11, 1 and 6 mJy in unconfused regions on the ecliptic in bands centered at wavelengths of 3.4, 4.6, 12 and 22 micrometers. Sensitivity improves toward the ecliptic poles due to denser coverage and lower zodiacal background. The angular resolution is 6.1", 6.4", 6.5" and 12.0" at 3.4, 4.6, 12 and 22 micrometers, and the astrometric precision for high SNR sources is better than 0.15"