The herpesvirus proteins HSV-1 ICP27 and HVS ORF57 promote viral mRNA export by utilizing the cellular mRNA export machinery. This function is triggered by binding to proteins of the transcription-export (TREX) complex, in particular to REF/Aly which directs viral mRNA to the TAP/NFX1 pathway and, subsequently, to the nuclear pore for export to the cytoplasm. Here we have determined the structure of the REF-ICP27 interaction interface at atomic-resolution and provided a detailed comparison of the binding interfaces between ICP27, ORF57 and REF using solution-state NMR. Despite the absence of any obvious sequence similarity, both viral proteins bind on the same site of the folded RRM domain of REF, via short but specific recognition sites. The regions of ICP27 and ORF57 involved in binding by REF have been mapped as residues 104–112 and 103–120, respectively. We have identified the pattern of residues critical for REF/Aly recognition, common to both ICP27 and ORF57. The importance of the key amino acid residues within these binding sites was confirmed by site-directed mutagenesis. The functional significance of the ORF57-REF/Aly interaction was also probed using an ex vivo cytoplasmic viral mRNA accumulation assay and this revealed that mutants that reduce the protein-protein interaction dramatically decrease the ability of ORF57 to mediate the nuclear export of intronless viral mRNA. Together these data precisely map amino acid residues responsible for the direct interactions between viral adaptors and cellular REF/Aly and provide the first molecular details of how herpes viruses access the cellular mRNA export pathway.

Bone morphogenetic proteins (BMPs) orchestrate key cellular events, such as proliferation and differentiation, in development and homeostasis. Extracellular antagonists, such as chordin, are essential regulators of BMP signaling. Chordin binds to BMPs blocking interaction with receptors, and cleavage by tolloid proteinases is thought to relieve this inhibition. A model has been previously proposed where chordin adopts a horseshoe-like arrangement enabling BMP binding cooperatively by terminal domains (1). Here, we present the nanoscale structure of human chordin using electron microscopy, small angle X-ray scattering, and solution-based biophysical techniques, which together show that chordin indeed has a compact horseshoe-shaped structure. Chordin variants were used to map domain locations within the chordin molecule. The terminal BMP-binding domains protrude as prongs from the main body of the chordin structure, where they are well positioned to interact with the growth factor. The spacing provided by the chordin domains supports the principle of a cooperative BMP-binding arrangement that the original model implied in which growth factors bind to both an N-and C-terminal von Willebrand factor C domain of chordin. Using binding and bioactivity assays, we compared full-length chordin with two truncated chordin variants, such as those produced by partial tolloid cleavage. Cleavage of either terminal domain has little effect on the affinity of chordin for BMP-4 and BMP-7 but C-terminal cleavage increases the efficacy of chordin as a BMP-4 inhibitor. Together these data suggest that partial tolloid cleavage is insufficient to ablate BMP inhibition and the C-terminal chordin domains play an important role in BMP regulation.

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The deconstruction of the plant cell wall is an important biological process that is attracting considerable industrial interest, particularly in the bioenergy sector. Enzymes that attack the plant cell wall generally contain one or more noncatalytic carbohydrate binding modules (CBMs) that play an important targeting function. While CBMs that bind to the backbones of plant structural polysaccharides have been widely described, modules that recognize components of the vast array of decorations displayed on these polymers have been relatively unexplored. Here we show that a family 35 CBM member (CBM35), designated CtCBM35-Gal, binds to alpha-D-galactose (Gal) and, within the context of the plant cell wall, targets the alpha-1,6-Gal residues of galactomannan but not the beta-D-Gal residues in xyloglucan. The crystal structure of CtCBM35-Gal reveals a canonical beta-sandwich fold. Site-directed mutagenesis studies showed that the ligand is accommodated within the loops that connect the two beta-sheets. Although the ligand binding site of the CBM displays significant structural similarity with calcium-dependent CBM35s that target uronic acids, subtle differences in the conformation of conserved residues in the ligand binding site lead to the loss of metal binding and uronate recognition. A model is proposed in which the orientation of the pair of aromatic residues that interact with the two faces of the Gal pyranose ring plays a pivotal role in orientating the axial O4 atom of the ligand toward Asn140, which is invariant in CBM35. The ligand recognition site of exo-CBM35s (CBM35-Gal and the uronic acid binding CBM35s) appears to overlap with that of CBM35-Man, which binds to the internal regions of mannan, a beta-polymer of mannose. Using site-directed mutagenesis, we show that although there is conservation of several functional residues within the binding sites of endo- and exo-CBM35s, the endo-CBM does not utilize Asn113 (equivalent to Asn140 in CBM35-Gal) in mannan binding, despite the importance of the equivalent residue in ligand recognition across the CBM35 and CBM6 landscape. The data presented in this report are placed within a wider phylogenetic context for the CBM35 family.

The solution structure of the GB1 domain of protein G at a pressure of 2 kbar is presented. The structure was calculated as a change from an energy-minimised low-pressure structure using (1)H chemical shifts. Two separate changes can be characterised: a compression/distortion, which is linear with pressure; and a stabilisation of an alternative folded state. On application of pressure, linear chemical shift changes reveal that the backbone structure changes by about 0.2 A root mean square, and is compressed by about 1% overall. The alpha-helix compresses, particularly at the C-terminal end, and moves toward the beta-sheet, while the beta-sheet is twisted, with the corners closest to the alpha-helix curling up towards it. The largest changes in structure are along the second beta-strand, which becomes more twisted. This strand is where the protein binds to IgG. Curved chemical shift changes with pressure indicate that high pressure also populates an alternative structure with a distortion towards the C-terminal end of the helix, which is likely to be caused by insertion of a water molecule.