Structural evidence for asymmetric ligand binding to transthyretin

Human transthyretin (TTR) represents a notable example of an amyloidogenic protein, and several compounds that are able to stabilize its native state have been proposed as effective drugs in the therapy of TTR amyloidosis. The two thyroxine (T4) binding sites present in the TTR tetramer display negative binding cooperativity. Here, structures of TTR in complex with three natural polyphenols (pterostilbene, quercetin and apigenin) have been determined, in which this asymmetry manifests itself as the presence of a main binding site with clear ligand occupancy and related electron density and a second minor site with a much lower ligand occupancy. The results of an analysis of the structural differences between the two binding sites are consistent with such a binding asymmetry. The different ability of TTR ligands to saturate the two T4 binding sites of the tetrameric protein can be ascribed to the different affinity of ligands for the weaker binding site. In comparison, the high-affinity ligand tafamidis, co-crystallized under the same experimental conditions, was able to fully saturate the two T4 binding sites. This asymmetry is characterized by the presence of small but significant differences in the conformation of the cavity of the two binding sites. Molecular-dynamics simulations suggest the presence of even larger differences in solution. Competition binding assays carried out in solution revealed the presence of a preferential binding site in TTR for the polyphenols pterostilbene and quercetin that was different from the preferential binding site for T4. The TTR binding asymmetry could possibly be exploited for the therapy of TTR amyloidosis by using a cocktail of two drugs, each of which exhibits preferential binding for a distinct binding site, thus favouring saturation of the tetrameric protein and consequently its stabilization.

Crystal structure of poly[(μ3-thiocyanato-κ3N:S:S)(trimethylphosphine sulfide-κS)copper(I)]

In the title compound, [Cu(NCS)(C3H9PS)]n, the thiocyanate ions bind the CuIatoms covalently, forming infinite –Cu—SCN—Cu– chains parallel to theaaxis. Each CuIatom is also coordinated to a trimethylphosphine sulfide groupviaa Cu—S bond. Two crystallographically independent chains propagate in opposite directions, and are held together in a ribbon arrangement by long bonds between CuIatoms in the first chain and thiocyanate S atoms in the second, with Cu—S = 2.621 (1) Å. The geometry around the CuIatoms in the first chain is distorted tetrahedral, with angles involving the long Cu—S bond much less than ideal, and the S—Cu—N angle between the phosphine sulfide S atom and the thiocyanate N atom opening out to 133.19 (9)°. Each CuIatom in the second chain appears to be disordered between two positions 0.524 (4) Å apart, with occupancy factors of 0.647 (6) and 0.353 (6). The CuIatom in the major site is in a distorted trigonal–planar configuration, with the S—Cu—N angle between the phosphine sulfide and the thiocyanate N atom again opened out, to 137.01 (15)°. The CuIatom in the minor site, however, forms in addition a long bond [Cu—S = 2.702 (5) Å] to the phosphine sulfide of the first chain, not the thiocyanate S atom, to provide a further link between the chains.

Importin alpha and nonclassical nuclear localization signal

In the classical nuclear import pathway, the specific recognition between the nuclear receptor (importin-α) and the nuclear localization signals (NLSs) plays an essential role on facilitating the cargo import process. Importin-α has two separate NLS-binding sites (the major and the minor sites), accommodate NLSs, comprising of one (monopartite) or two clusters (bipartite) of basic residues connected by a 10 - 12 residue linker. The major NLS-binding site is the preferential binding site for most of the monopartite NLSs characterized to date. By screening random peptide libraries using importin-α variants as bait, the bound NLS sequences could be divided into six classes [1]. The class-3 minor site-specific NLSs and class-5 plant-specific NLSs feature a shorter basic cluster. The molecular basis of the specific binding between these non-classical NLSs and importin-α was not known and in particular, there was a lack of crystal structures of plant importin-α. Here, we present the first crystal structure of plant importin-α, and explain the differential binding specificity between the class-5 plant-specific NLSs and importin-α variants [2]. The binding conformation of the class-3 minor site-specific NLSs features an α-helical turn, that is distinct from the other NLSs reported structurally [3]. Comparative bioinformatic screens not only indicate both plant-specific and minor site-specific NLSs are much less prevalent than the classical NLSs, but also reveal a greater prevalence of these two classes of non-classical NLSs in rice the proteome, compared to the others from yeast, mammals, and even other plant species. Together, our data can help to characterize novel proteins containing non-classical NLSs destined for the cell nucleus by the classical nuclear import pathway.

Energetics and Location of Phosphoinositide Binding in Human Kir2.1 Channels

Kir2.1 channels are uniquely activated by phosphoinositide 4,5-bisphosphate (PI(4,5)P2) and can be inhibited by other phosphoinositides (PIPs). Using biochemical and computational approaches, we assess PIP-channel interactions and distinguish residues that are energetically critical for binding from those that alter PIP sensitivity by shifting the open-closed equilibrium. Intriguingly, binding of each PIP is disrupted by a different subset of mutations. In silico ligand docking indicates that PIPs bind to two sites. The second minor site may correspond to the secondary anionic phospholipid site required for channel activation. However, 96–99% of PIP binding localizes to the first cluster, which corresponds to the general PI(4,5)P2 binding location in recent Kir crystal structures. PIPs can encompass multiple orientations; each di- and triphosphorylated species binds with comparable energies and is favored over monophosphorylated PIPs. The data suggest that selective activation by PI(4,5)P2 involves orientational specificity and that other PIPs inhibit this activation through direct competition.

Architecture of the Sporulation-Specific Cdc14 Promoter from the Oomycete Phytophthora infestans

ABSTRACT The Cdc14 gene of Phytophthora infestans is transcribed specifically during sporulation, with no mRNA detectable in vegetative hyphae, and is required for sporangium development. To unravel the mechanisms regulating its transcription, mutated Cdc14 promoters plus chimeras of selected Cdc14 sequences and a minimal promoter were tested in stable transformants. This revealed that a tandem repeat of three copies of the motif CTYAAC, located between 67 and 90 nucleotides (nt) upstream of the major transcription start site, is sufficient to determine sporulation-specific expression. All three repeats need to be present for activity, suggesting that they bind a transcription factor through a cooperative mechanism. Electrophoretic mobility shift assays indicated that the CTYAAC repeats are specifically bound by a protein in nuclear extracts. Evidence was also obtained for a second region within the promoter that activates Cdc14 transcription during sporulation which does not involve those repeats. The CTYAAC motif also affects the specificity of transcription initiation. Wild-type Cdc14 is transcribed from a major start site and minor site(s) located about 100 nt upstream of the major site. However, stepwise mutations through the CTYAAC triad caused a graded shift to the upstream sites, as did mutating bases surrounding the major start site; transcripts initiated from the upstream site remained sporulation specific. Replacing the Cdc14 initiation region with the Inr-like region of the constitutive Piexo1 gene had no apparent effect on the pattern of transcription. Therefore, this study reports the first motif determining sporulation-induced transcription in oomycetes and helps define oomycete core promoters.