Structural features of the scaffold interaction domain at the N terminus of the major capsid protein (VP5) of herpes simplex virus type 1

Protein-protein interactions drive the assembly of the herpes simplex virus type 1 capsid. A key interaction occurs between the C terminus of the scaffold protein and the N terminus of the major capsid protein (VP5). Results from alanine-scanning mutagenesis of hydrophobic residues in the N terminus of VP5 revealed seven residues (I27, L35, F39, L58, L65, L67, and L71) that reside in two predicted alpha helices (helix 1(22-42) and helix 2(58-72)) that are important for this bimolecular interaction. The goal of the present study was to further characterize the VP5 scaffold interaction domain (SID). Amino acids at the seven positions were replaced with L, M, V or P (I27); I, M, V, or P (L35, L58, L65, L67, and L71); and H, W, Y, or L (F39). Replacement with a hydrophobic side chain did not affect the interaction with scaffold protein in yeast cells or the ability of a virus specifying the mutation from replicating in cells. The mutation to the proline side chain abolished the interaction in all cases and was lethal for virus replication. Mutant viruses with proline substitutions in helix 1(22-42) at positions 27 and 35 assembled large open capsid shells that did not attain closure. Proline substitutions in helix 2(58-72) at either position 59, 65, or 67 abolished the accumulation of VP5 protein, and, at 58 and 71, although VP5 did accumulate, capsid shells were not assembled. Thus, the second SID, SID2, is highly structured, and this alpha helix (helix 2(58-72)) is likely involved in capsomere-capsomere interactions during shell accretion. Conserved glycine G59 in helix 2(58-72) was also mutated. G59 may act as a flexible "hinge" in helix 2(58-72) because decreasing the movement of this side chain by replacement with valine impaired capsid assembly. Thus, the N terminus of VP5 and the alpha helices embedded in this domain, as in the capsid shell proteins of some double-stranded DNA phages, are a key regulator of shell accretion and stabilization.     

Pleiotropic corepressors Sin3 and Ssn6 interact with repressor Opi1 and negatively regulate transcription of genes required for phospholipid biosynthesis in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Repressor protein Opi1 is required to negatively regulate yeast structural genes of phospholipid biosynthesis in the presence of precursor molecules inositol and choline (IC). Opi1 interacts with the paired amphipathic helix 1 (PAH1) of pleiotropic corepressor Sin3, leading to recruitment of histone deacetylases (HDACs). Mutational analysis of the Opi1–Sin3 interaction domain (OSID) revealed that hydrophobic OSID residues L56, V59 and V67 of Opi1 are indispensable for gene repression. Our results also suggested that repression is not executed entirely via Sin3. Indeed, we could show that OSID contacts a second pleiotropic corepressor, Ssn6 (=Cyc8), which together with Tup1 is also able to recruit HDACs. Interestingly, mutations sin3 and ssn6 turned out as synthetically lethal. Our analysis further revealed that OSID not only binds to PAH1 but also interacts with tetratricopeptide repeats (TPR) of Ssn6. This interaction could no longer be observed with Opi1 OSID variants. To trigger gene repression, Opi1 must also interact with activator Ino2, using its activator interaction domain (AID). AID contains a hydrophobic structural motif reminiscent of a leucine zipper. Our mutational analysis of selected positions indeed confirmed that residues L333, L340, V343, V350, L354 and V361 are necessary for repression of Opi1 target genes.     

Crystal structure of the GAF-B domain from human phosphodiesterase 10A complexed with its ligand, cAMP

Cyclic nucleotide phosphodiesterases (PDEs) catalyze the degradation of the cyclic nucleotides cAMP and cGMP, which are important second messengers. Five of the 11 mammalian PDE families have tandem GAF domains at their N termini. PDE10A may be the only mammalian PDE for which cAMP is the GAF domain ligand, and it may be allosterically stimulated by cAMP. PDE10A is highly expressed in striatal medium spiny neurons. Here we report the crystal structure of the C-terminal GAF domain (GAF-B) of human PDE10A complexed with cAMP at 2.1-angstroms resolution. The conformation of the PDE10A GAF-B domain monomer closely resembles those of the GAF domains of PDE2A and the cyanobacterium Anabaena cyaB2 adenylyl cyclase, except for the helical bundle consisting of alpha1, alpha2, and alpha5. The PDE10A GAF-B domain forms a dimer in the crystal and in solution. The dimerization is mainly mediated by hydrophobic interactions between the helical bundles in a parallel arrangement, with a large buried surface area. In the PDE10A GAF-B domain, cAMP tightly binds to a cNMP-binding pocket. The residues in the alpha3 and alpha4 helices, the beta6 strand, the loop between 3(10) and alpha4, and the loop between alpha4 and beta5 are involved in the recognition of the phosphate and ribose moieties. This recognition mode is similar to those of the GAF domains of PDE2A and cyaB2. In contrast, the adenine base is specifically recognized by the PDE10A GAF-B domain in a unique manner, through residues in the beta1 and beta2 strands.