Background: The evolution of the plant xyloglucan endotransglycosylase/hydrolase (XTH) genes in glycoside hydrolase family 16 (GH16) is enigmatic. Results: A unique, mixed function endo(xylo)glucanase from black cottonwood has been biochemically and structurally characterized. Conclusion:This enzyme is an important link between extant bacterial endoglucanases and plant XTH gene products. Significance: New insights into the molecular evolution of XTH gene products and further unification of GH16 enzymes have been gained.

Human immunodeficiency virus type 1 (HIV-1) 2 -infected resting CD4 ϩ memory T-cells form a persistent viral reservoir that is a major barrier to curing HIV-1 infection although they do not permit viral replication (1). A small, RNA-binding protein, Tat (transactivator of transcription) (2), plays a central role in the regulation of HIV-1 replication and in potential approaches to treating latently infected cells. During transcription of the viral DNA, RNA polymerase II stalls as a result of binding the negative transcription elongation factor, resulting in the production of prematurely terminated transcripts that may include the tat message (3). Following translation, the Tat protein is transported from the cytoplasm into the nucleus, where it binds a stem-loop structure (transactivation response element (TAR)) formed by the first 59 nucleotides of the HIV-1 RNA (4). Tat stimulates elongation of full-length transcripts by recruiting the positive transcription elongation factor b, a complex of a regulatory cyclin and cyclin-dependent kinase 9, that phosphorylates the C-terminal domain of RNA polymerase II, components of negative transcription elongation factor, and the transcription elongation factor Spt5 (5-7). Recent results suggest that Tat activates positive transcription elongation factor b by displacing Hexim1 (hexamethylene bisacetamide-inducible protein 1) from its cyclin T1 binding site (8) and that the affinity of the Tat-cyclin T1-cyclindependent kinase 9 complex for TAR is regulated through Tat acetylation by histone acetyltransferases (9, 10). The absence of Tat and low levels of cyclin-dependent kinase 9 and cyclin T1 in resting CD4ϩ T-cells are both implicated in HIV-1 latency (1). Tat may also be involved in derepression of heterochromatin, transcription initiation (11), and reverse transcription (12). In addition to its intracellular activities, the literature contains a plethora of reports of extracellular Tat activities, including general cytotoxicity, that may contribute to immune suppression (13,14) and the development of HIV-dementia (15). A molecular understanding of Tat activity requires a determination of its structure and interactions with cellular and viral partners (16, 17). HIV-1 Tat is a 101-residue protein encoded by two exons (3, 18). The first exon defines amino acids 1-72 that encompass an acidic and proline-rich N terminus (amino acids 1-21), a cysteine-rich region (amino acids 22-37), a core (amino acids 38 -47), a basic region (amino acids 48 -57), and a Gln-rich segment (amino acids 58 -72) (19); it activates transcription with the same proficiency as the full-length protein (3, 20 -22). Residues 1-24 form the co-activator and acetyltransferase CREB-binding protein KIX domain binding site (17). Cyclin T1 is thought to interact with the Cys-rich region of Tat (23), and mutation of any one of six of the seven Cys residues results in inactivation (3). The end of the Cys-rich region and the core are involved in mitochondrial apoptosis of bystander noninfected cells through thei...

SH2 domain-containing inositol-5'-phosphatase-1 (SHIP1) inhibits inflammation by hydrolyzing phosphoinositide-3'-kinase generated membrane phosphatidylinositol-3,4,5-trisphosphate (PIP(3)). Bioinformatic analysis of SHIP1 from multiple species revealed a pleckstrin homololgy-related (PH-R) domain, which we hypothesize mediates SHIP1's association with the membrane, a requirement for its biological function. Recombinant murine SHIP1 PH-R domain was subjected to biophysical and biochemical analysis. Residues K370 and K397 were found to be important for PH-R domain association with membrane PIP(3). Wild-type PH-R domain bound PIP(3) with 1.9 ± 0.2 nM affinity, while the affinity of a K370A/K397A substituted mutant was too low to measure. Wild-type (but not the K370A/K397A substituted) full-length SHIP1 protein, reconstitutes normal inhibition of Fcγ receptor-mediated phagocytosis when introduced into SHIP1(-/-) murine macrophages, reducing the number of phagocytic events by 2-fold as compared to SHIP1(-/-) cells. In fact, the PH-R-mediated membrane interaction appears to be a major mechanism by which SHIP1 is recruited to the membrane, since the K370A/K397A substitution reduced the recruitment of both full-length SHIP1 and the PH-R domain by ≥2-fold. We have previously shown that SHIP1 enzyme activity can be targeted for therapeutic purposes. The current studies suggest that molecules targeting the PH-R domain can also modulate SHIP1 function.

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The type 1 Human Immunodeficiency Virus transcriptional regulator Tat is a small RNA-binding protein essential for viral gene expression and replication. The protein binds to a large number of proteins within infected cells and non-infected cells, and has been demonstrated to impact a wide variety of cellular activities. Early circular dichroism studies showed a lack of regular secondary structure in the protein whereas proton NMR studies suggested several different conformations. Multinuclear NMR structure and dynamics analysis indicates that the reduced protein is intrinsically disordered with a predominantly extended conformation at pH 4. Multiple resonances for several atoms suggest the existence of multiple local conformers in rapid equilibrium. An X-ray diffraction structure of equine Tat, in a complex with its cognate RNA and cyclin T1, supports this conclusion. Intrinsic disorder explains the protein's capacity to interact with multiple partners and effect multiple biological functions; the large buried surface in the X-ray diffraction structure illustrates how a disordered protein can have a high affinity and high specificity for its partners and how disordered Tat assembles a protein complex to enhance transcription elongation.

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The human immunodeficiency virus type 1 (HIV-1) genome encodes 18 proteins and 2 peptides. Four of these proteins encode high-affinity calmodulin-binding sites for which direct interactions with calmodulin have already been described. In this study, the HIV-1 proteome is queried using an algorithm that predicts calmodulin-binding sites revealing seven new putative calmodulin-binding sites including residues 34-56 of the transactivator of transcription (Tat). Tat is a 101-residue intrinsically disordered RNA-binding protein that plays a central role in the regulation of HIV-1 replication. Interactions between a Tat peptide (residues 34-56), melittin, a well-characterized calmodulin-binding peptide, and calmodulin were examined by direct binding studies, mass spectrometry, and fluorescence. The Tat peptide binds to both calcium-saturated and apo-calmodulin with a low micromolar affinity. Conformational changes induced in the Tat peptide were determined by circular dichroism, and residues in calmodulin that interact with the peptide were identified by HSQC NMR spectroscopy. Multiple interactions between HIV-1 proteins and calmodulin, a highly promiscuous signal transduction hub protein, may be an important mechanism by which the virus controls cell physiology.

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