In Human Pseudouridine Synthase 1 (hPus1), a C-Terminal Helical Insert Blocks tRNA from Binding in the Same Orientation as in the Pus1 Bacterial Homologue TruA,Consistent with Their Different Target Selectivities

Human pseudouridine (Ψ) synthase Pus1 (hPus1) modifies specific uridine residues in several non-coding RNAs: tRNA, U2 spliceosomal RNA, and steroid receptor activator RNA. We report three structures of the catalytic core domain of hPus1 from two crystal forms, at 1.8 Å resolution. The structures are the first of a mammalian Ψ synthase from the set of five Ψ synthase families common to all kingdoms of life. hPus1 adopts a fold similar to bacterial Ψ synthases, with a central antiparallel β-sheet flanked by helices and loops. A flexible hinge at the base of the sheet allows the enzyme to open and close around an electropositive active-site cleft. In one crystal form, a molecule of Mes [2-(N-morpholino)ethane sulfonic acid] mimics the target uridine of an RNA substrate. A positively charged electrostatic surface extends from the active site towards the N-terminus of the catalytic domain, suggesting an extensive binding site specific for target RNAs. Two α-helices C-terminal to the core domain, but unique to hPus1, extend along the back and top of the central β-sheet and form the walls of the RNA binding surface. Docking of tRNA to hPus1 in a productive orientation requires only minor conformational changes to enzyme and tRNA. The docked tRNA is bound by the electropositive surface of the protein employing a completely different binding mode than that seen for the tRNA complex of the Escherichia coli homologue TruA.     

The Four Canonical TPR Subunits of Human APC/C Form Related Homo-Dimeric Structures and Stack in Parallel to Form a TPR Suprahelix

The anaphase-promoting complex or cyclosome (APC/C) is a large E3 RING-cullin ubiquitin ligase composed of between 14 and 15 individual proteins. A striking feature of the APC/C is that only four proteins are involved in directly recognizing target proteins and catalyzing the assembly of a polyubiquitin chain. All other subunits, which account for > 80% of the mass of the APC/C, provide scaffolding functions. A major proportion of these scaffolding subunits are structurally related. In metazoans, there are four canonical tetratricopeptide repeat (TPR) proteins that form homo-dimers (Apc3/Cdc27, Apc6/Cdc16, Apc7 and Apc8/Cdc23). Here, we describe the crystal structure of the N-terminal homo-dimerization domain of Schizosaccharomyces pombe Cdc23 (Cdc23^Nterm). Cdc23^Nterm is composed of seven contiguous TPR motifs that self-associate through a related mechanism to those of Cdc16 and Cdc27. Using the Cdc23^Nterm structure, we generated a model of full-length Cdc23. The resultant “V”-shaped molecule docks into the Cdc23-assigned density of the human APC/C structure determined using negative stain electron microscopy (EM). Based on sequence conservation, we propose that Apc7 forms a homo-dimeric structure equivalent to those of Cdc16, Cdc23 and Cdc27. The model is consistent with the Apc7-assigned density of the human APC/C EM structure. The four canonical homo-dimeric TPR proteins of human APC/C stack in parallel on one side of the complex. Remarkably, the uniform relative packing of neighboring TPR proteins generates a novel left-handed suprahelical TPR assembly. This finding has implications for understanding the assembly of other TPR-containing multimeric complexes.     

Low-Molecular-Weight DNA Replication Intermediates in Escherichia coli: Mechanism of Formation and Strand Specificity

Chromosomal DNA replication intermediates, revealed in ligase-deficient conditions in vivo, are of low molecular weight (LMW) independently of the organism, suggesting discontinuous replication of both the leading and the lagging DNA strands. Yet, in vitro experiments with purified enzymes replicating sigma-structured substrates show continuous synthesis of the leading DNA strand in complete absence of ligase, supporting the textbook model of semi-discontinuous DNA replication. The discrepancy between the in vivo and in vitro results is rationalized by proposing that various excision repair events nick continuously synthesized leading strands after synthesis, producing the observed LMW intermediates. Here, we show that, in an Escherichia coli ligase-deficient strain with all known excision repair pathways inactivated, new DNA is still synthesized discontinuously. Furthermore, hybridization to strand-specific targets demonstrates that the LMW replication intermediates come from both the lagging and the leading strands. These results support the model of discontinuous leading strand synthesis in E. coli.     

Acyl Transfer from Membrane Lipids to Peptides Is a Generic Process

The generality of acyl transfer from phospholipids to membrane-active peptides has been probed using liquid chromatography–mass spectrometry analysis of peptide–lipid mixtures. The peptides examined include melittin, magainin II, PGLa, LAK1, LAK3 and penetratin. Peptides were added to liposomes with membrane lipid compositions ranging from pure phosphatidylcholine (PC) to mixtures of PC with phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol. Experiments were typically conducted at pH 7.4 at modest salt concentrations (90 mM NaCl). In favorable cases, lipidated peptides were further characterized by tandem mass spectrometry methods to determine the sites of acylation. Melittin and magainin II were the most reactive peptides, with significant acyl transfer detected under all conditions and membrane compositions. Both peptides were lipidated at the N-terminus by transfer from PC, phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol, as well as at internal sites: lysine for melittin; serine and lysine for magainin II. Acyl transfer could be detected within 3 h of melittin addition to negatively charged membranes. The other peptides were less reactive, but for each peptide, acylation was found to occur in at least one of the conditions examined. The data demonstrate that acyl transfer is a generic process for peptides bound to membranes composed of diacylglycerophospholipids. Phospholipid membranes cannot therefore be considered as chemically inert toward peptides and by extension proteins.     

Effect of C-Terminal Sequence on Competitive Semaphorin Binding to Neuropilin-1

Neuropilins (Nrp) are type I transmembrane proteins that function as receptors for vascular endothelial growth factor (VEGF) and class III Semaphorin (Sema3) ligand families. Sema3s function as potent endogenous angiogenesis inhibitors but require proteolytically processing by furin to compete with VEGF for Nrp binding. This processing liberates a C-terminal arginine (C_R) that is necessary for binding to the b1 domain of Nrp, a common feature shared by Nrp ligands. The C_R is necessary but not sufficient for potent Nrp inhibition, and the role of upstream residues is unknown. We demonstrate that the second-to-last residue (C-1), immediately upstream of the C_R, plays a significant role in controlling competitive ligand binding by orienting the C-terminus for productive Nrp binding. With the use of a peptide library derived from Sema3F, C-1 residues that preferentially adopt an extended bound-like conformation, including proline and β-branched amino acids, were found to produce the most avid competitors. Consistent with this, analysis of the binding thermodynamics revealed that more favorable entropy is responsible for the observed binding enhancement of C-1 proline. We further tested the effect of the C-1 residue on Sema3F processing by furin and found an inverse relationship between processing and inhibitory potency. Analysis of all Sema3 family members reveals two non-equivalent furin processing sites differentiated by the presence of either a C-1 proline or a C-1 arginine and resulting in up to a 40-fold difference in potency. These data reveal a novel regulatory mechanism of Sema3 activity and define a fundamental mechanism for preferential Nrp binding.     

Structural Mechanism of Replication Stalling on a Bulky Amino-Polycyclic Aromatic Hydrocarbon DNA Adduct by a Y Family DNA Polymerase

Polycyclic aromatic hydrocarbons and their nitro derivatives are culprits of the detrimental health effects of environmental pollution. These hydrophobic compounds metabolize to reactive species and attach to DNA producing bulky lesions, such as N-[deoxyguanosine-8-yl]-1-aminopyrene (APG), in genomic DNA. The bulky adducts block DNA replication by high-fidelity polymerases and compromise replication fidelities and efficiencies by specialized lesion bypass polymerases. Here we present three crystal structures of the DNA polymerase Dpo4, a model translesion DNA polymerase of the Y family, in complex with APG-lesion-containing DNA in pre-insertion and extension stages. APG is captured in two conformations in the pre-insertion complex; one is highly exposed to the solvent, whereas the other is harbored in a shallow cleft between the finger and unique Y family little finger domain. In contrast, APG is in a single conformation at the extension stage, in which the pyrene ring is sandwiched between the little finger domain and a base from the turning back single-stranded template strand. Strikingly, a nucleotide intercalates the DNA helix to form a quaternary complex with Dpo4, DNA, and an incoming nucleotide, which stabilizes the distorted DNA structure at the extension stage. The unique APG DNA conformations in Dpo4 inhibit DNA translocation through the polymerase active site for APG bypass. We also modeled an insertion complex that illustrates a solvent-exposed pyrene ring contributing to an unstable insertion state. The structural work combined with our lesion replication assays provides a novel structural mechanism on bypass of DNA adducts containing polycyclic aromatic hydrocarbon moieties.     

Nucleotide-Specific Recognition of Iron-Responsive Elements by Iron Regulatory Protein 1

IRP1 [iron regulatory protein (IRP) 1] is a bifunctional protein with mutually exclusive end-states. In one mode of operation, IRP1 binds iron-responsive element (IRE) stem–loops in messenger RNAs encoding proteins of iron metabolism to control their rate of translation. In its other mode, IRP1 serves as cytoplasmic aconitase to correlate iron availability with the energy and oxidative stress status of the cell. IRP1/IRE binding occurs through two separate interfaces, which together contribute about two-dozen hydrogen bonds. Five amino acids make base-specific contacts and are expected to contribute significantly to binding affinity and specificity of this protein:RNA interaction. In this mutagenesis study, each of the five base-specific amino acids was changed to alter binding at each site. Analysis of IRE binding affinity and translational repression activity of the resulting IRP1 mutants showed that four of the five contact points contribute uniquely to the overall binding affinity of the IRP1:IRE interaction, while one site was found to be unimportant. The stronger-than-expected effect on binding affinity of mutations at Lys379 and Ser681, residues that make contact with the conserved nucleotides G16 and C8, respectively, identified them as particularly critical for providing specificity and stability to IRP1:IRE complex formation. We also show that even though the base-specific RNA-binding residues are not part of the aconitase active site, their substitutions can affect the aconitase activity of holo-IRP1, positively or negatively.