Structural insights into TAZ2 domain-mediated CBP/p300 recruitment by transactivation domain 1 of the lymphopoietic transcription factor E2A.

The E-protein transcription factors guide immune cell differentiation, with E12 and E47 (hereafter called E2A) being essential for B-cell specification and maturation. E2A and the oncogenic chimera E2A-PBX1 contain three transactivation domains (ADs), with AD1 and AD2 having redundant, independent, and cooperative functions in a cell-dependent manner. AD1 and AD2 both mediate their functions by binding to the KIX domain of the histone acetyltransferase paralogues CREB-binding protein (CBP) and E1A-binding protein P300 (p300). This interaction is necessary for B-cell maturation and oncogenesis by E2A-PBX1 and occurs through conserved ϕ-x-x-ϕ-ϕ motifs (with ϕ denoting a hydrophobic amino acid) in AD1 and AD2. However, disruption of this interaction via mutation of the KIX domain in CBP/p300 does not completely abrogate binding of E2A and E2APBX1. Here, we determined that E2A-AD1 and E2A-AD2 also interact with the TAZ2 domain of CBP/p300. Characterization of the TAZ2:E2AAD1(1-37) complex indicated that E2A-AD1 adopts an α-helical structure and uses its ϕ-x-x-ϕ-ϕ motif to bind TAZ2. While this region overlapped with the KIX recognition region, key KIX-interacting E2A-AD1 residues were exposed, suggesting that E2A-AD1 could simultaneously bind both the KIX and TAZ2 domains. However, we did not detect a ternary complex involving E2A-AD1, KIX, and TAZ2 and found that E2A containing both intact AD1 and AD2 is required to bind to CBP/p300. Our findings highlight the structural plasticity and promiscuity of E2A-AD1 and suggest that E2A binds both the TAZ2 and KIX domains of CBP/p300 through AD1 and AD2

Glucose Monitoring During Pregnancy

Self-monitoring of blood glucose in women with mild gestational diabetes has recently been proven to be useful in reducing the rates of fetal overgrowth and gestational weight gain. However, uncertainty remains with respect to the optimal frequency and timing of self-monitoring. A continuous glucose monitoring system may have utility in pregnant women with insulin-treated diabetes, especially for those women with blood sugars that are difficult to control or who experience nocturnal hypoglycemia; however, continuous glucose monitoring systems need additional study as part of larger, randomized trials

Diverse modes of galacto-specific carbohydrate recognition by a family 31 glycoside hydrolase from <i>Clostridium perfringens</i>

<div><p><i>Clostridium perfringens</i> is a commensal member of the human gut microbiome and an opportunistic pathogen whose genome encodes a suite of putative large, multi-modular carbohydrate-active enzymes that appears to play a role in the interaction of the bacterium with mucin-based carbohydrates. Among the most complex of these is an enzyme that contains a presumed catalytic module belonging to glycoside hydrolase family 31 (GH31). This large enzyme, which based on its possession of a GH31 module is a predicted α-glucosidase, contains a variety of non-catalytic ancillary modules, including three CBM32 modules that to date have not been characterized. NMR-based experiments demonstrated a preference of each module for galacto-configured sugars, including the ability of all three CBM32s to recognize the common mucin monosaccharide GalNAc. X-ray crystal structures of the <i>Cp</i>GH31 CBM32s, both in apo form and bound to GalNAc, revealed the finely-tuned molecular strategies employed by these sequentially variable CBM32s in coordinating a common ligand. The data highlight that sequence similarities to previously characterized CBMs alone are insufficient for identifying the molecular mechanism of ligand binding by individual CBMs. Furthermore, the overlapping ligand binding profiles of the three CBMs provide a fail-safe mechanism for the recognition of GalNAc among the dense eukaryotic carbohydrate networks of the colonic mucosa. These findings expand our understanding of ligand targeting by large, multi-modular carbohydrate-active enzymes, and offer unique insights into of the expanding ligand-binding preferences and binding site topologies observed in CBM32s.</p></div

Carbohydrate binding preferences of the <i>Cp</i>GH31 CBM32 modules.

<p>Regions for the two-dimensional ¹H-¹⁵N HSQC spectra of 100 μM CBM32-1, 184 μM CBM32-2, and 100 μM CBM32-3 at pH 6.9 in the absence (black) and presence (red) of 8 mM galactose (Gal), glucose (Glc), N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), N-acetyllactosamine (LacNAc) or N-glucosamine (GlcN).</p

Oligonucleotide primers used for cloning of the <i>Cp</i>GH31 CBM32s.

<p>Oligonucleotide primers used for cloning of the <i>Cp</i>GH31 CBM32s.</p

Amino acid sequence comparison of GalNAc-binding <i>C</i>. <i>perfringens</i> CBM32s.

<p>Sequence alignment of the three <i>Cp</i>GH31 CBM32 modules with other functionally characterized CBM32 modules from the following family 33, family 84, and family 89 glycoside hydrolases with specificity for galacto- or gluco-configured sugars: <i>Cp</i>GH33 CBM32, galacto-configured sugar specificity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref021" target="_blank">21</a>]; <i>Cp</i>GH84A CBM32-1, galacto-configured sugar specificity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref024" target="_blank">24</a>]; <i>Cp</i>GH84A CBM32-2, GlcNAc specificity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref023" target="_blank">23</a>]; <i>Cp</i>GH84C CBM32, galacto-configured sugar specificity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref022" target="_blank">22</a>]; <i>Cp</i>GH89 CBM32-3 and CBM32-4, GlcNAc-α-1,4-Gal specificity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref020" target="_blank">20</a>]; <i>Cp</i>GH89 CBM32-5, galacto-configured sugar specificity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref020" target="_blank">20</a>]. Positions comprising conserved amino acid residues are identified by white single-letter code and highlighted in red while positions displaying amino acid residues of similar physicochemical properties are identified by red-single letter code. Sugar-coordinating amino acid residues in each CBM32 seequence are identified by black boxes. The sequence alignment was created using CLUSTAL OMEGA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref045" target="_blank">45</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref046" target="_blank">46</a>] and displayed using ESPript [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref047" target="_blank">47</a>].</p

Comparison of the GalNAc binding sites of the <i>Cp</i>GH31 CBM32s.

<p>The variable loop regions of (A) CBM32-1 (red), (B) CBM32-2 (orange), and (C) CBM32-3 (violet) contain a similar complement of residues involved in the recognition of GalNAc (green), including one or more aromatic residues. The structural conservation of key residues in the variable loop regions of CBM32-1 and CBM32-3 (His990, Phe1085, Tyr972 and His1671, Tyr1774, Tyr1674, respectively) allowed for GalNAc to be modeled into the binding site of CBM32-1. Specifically, <i>Cp</i>GH31 CBM32-3:GalNAc was identified as the top structural homologue of CBM32-1 using the DALI server (Z-score of 18.1; backbone r.s.m.d. of 1.9 Å), and the two structures were superimposed in order to position GalNAc into the binding site of CBM32-1. The binding site of CBM32-2 is unique as these residues are not conserved, but rather replaced by an extensive suite of residues involved in the coordination of GalNAc via hydrogen bonding. Residues are represented by their single-letter amino acid code. (D) Amino acid sequence alignment of the three <i>Cp</i>GH31 modules. Positions comprising conserved amino acid residues are identified by white single-letter code and highlighted in red while positions displaying amino acid residues of similar physicochemical properties are identified by red-single letter code. An asterisk denotes amino acid residues of <i>Cp</i>GH31 CBM32-1 implicated in GalNAc recognition by NMR titrations while those coordinating GalNAc in the <i>Cp</i>GH31 CBM32-2 and CBM32-3 are identified by pound and ampersand symbols, respectively. The sequence alignment was created using CLUSTAL OMEGA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref045" target="_blank">45</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref046" target="_blank">46</a>] and displayed using ESPript [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref047" target="_blank">47</a>].</p