H. van Tilbeurgh

Colipase: structure and interaction with pancreatic lipase

Colipase is a small protein cofactor needed by pancreatic lipase for the efficient dietary lipid hydrolysis. It binds to the C-terminal, non-catalytic domain of lipase, thereby stabilising an active conformation and considerably increasing the overall hydrophobic binding site. Structural studies of the complex and of colipase alone have clearly revealed the functionality of its architecture. Interestingly, a structural analogy has recently been discovered between colipase and a domain in a developmental protein (Dickkopf), based on sequence analogy and homology modeling. Whether this structural analogy implies a common function (lipid interaction) remains to be clarified. Structural analogies have also been recognised between the pancreatic lipase C-terminal domain, the N-terminal domains of lipoxygenases and the C-terminal domain of alpha-toxin. These non-catalytic domains in the latter enzymes are important for interaction with membranes. It has not been established if these domains are also involved in eventual protein cofactor binding as is the case for pancreatic lipase.

Functional and Structural Analysis of HicA3-HicB3, a Novel Toxin-Antitoxin System of Yersinia pestis

The mechanisms involved in the virulence of Yersinia pestis, the plague pathogen, are not fully understood. In previous research, we found that a Y. pestis mutant lacking the HicB3 (YPO3369) putative orphan antitoxin was attenuated for virulence in a murine model of bubonic plague. Toxin-antitoxin systems (TASs) are widespread in prokaryotes. Most bacterial species possess many TASs of several types. In type II TASs, the toxin protein is bound and neutralized by its cognate antitoxin protein in the cytoplasm. Here we identify the hicA3 gene encoding the toxin neutralized by HicB3 and show that HicA3-HicB3 constitutes a new functional type II TAS in Y. pestis. Using biochemical and mutagenesis-based approaches, we demonstrate that the HicA3 toxin is an RNase with a catalytic histidine residue. HicB3 has two functions: it sequesters and neutralizes HicA3 by blocking its active site, and it represses transcription of the hicA3B3 operon. Gel shift assays and reporter fusion experiments indicate that the HicB3 antitoxin binds to two operators in the hicA3B3 promoter region. We solved the X-ray structures of HicB3 and the HicA3-HicB3 complex; thus, we present the first crystal structure of a TA complex from the HicAB family. HicB3 forms a tetramer that can bind two HicA3 toxin molecules. HicA3 is monomeric and folds as a double-stranded-RNA-binding domain. The HicB3 N-terminal domain occludes the HicA3 active site, whereas its C-terminal domain folds as a ribbon-helix-helix DNA-binding motif.

A protein encoded by a new family of mobile elements from Euryarchaea exhibits three domains with novel folds

We present here the 2.6Å resolution crystal structure of the pT26‐6p protein, which is encoded by an ORF of the plasmid pT26‐2, recently isolated from the hyperthermophilic archaeon, Thermococcus sp. 26,2. This large protein is present in all members of a new family of mobile elements that, beside pT26‐2 include several virus‐like elements integrated in the genomes of several Thermococcales and Methanococcales (phylum Euryarchaeota). Phylogenetic analysis suggested that this protein, together with its nearest neighbor (organized as an operon) have coevolved for a long time with the cellular hosts of the encoding mobile element. As the sequences of the N and C‐terminal regions suggested a possible membrane association, a deletion construct (739 amino acids) was used for structural analysis. The structure consists of two very similar β‐sheet domains with a new topology and a five helical bundle C‐terminal domain. Each of these domains corresponds to a unique fold that has presently not been found in cellular proteins. This result supports the idea that proteins encoded by plasmid and viruses that have no cellular homologues could be a reservoir of new folds for structural genomic studies.

High-throughput crystal-optimization strategies in the South Paris Yeast Structural Genomics Project: one size fits all?

Crystallization has long been regarded as one of the major bottlenecks in high-throughput structural determination by X-ray crystallography. Structural genomics projects have addressed this issue by using robots to set up automated crystal screens using nanodrop technology. This has moved the bottleneck from obtaining the first crystal hit to obtaining diffraction-quality crystals, as crystal optimization is a notoriously slow process that is difficult to automatize. This article describes the high-throughput optimization strategies used in the Yeast Structural Genomics project, with selected successful examples.

A Novel Protein-Protein Interaction in the Res (Retention and Splicing) Complex.

Background: The heterotrimeric retention and splicing (RES) complex is conserved from yeast to man. Results: Structural and biochemical analyses show an extended binding interface between the Snu17p RNA recognition motif (RRM) and Bud13p of the RES complex. Conclusion: The Snu17p RRM-Bud13p interaction represents a novel RRM-protein interface. Significance: The Snu17p-Bud13p complex may provide a platform for additional protein and RNA interactions in the RES complex. The retention and splicing (RES) complex is a conserved spliceosome-associated module that was shown to enhance splicing of a subset of transcripts and promote the nuclear retention of unspliced pre-mRNAs in yeast. The heterotrimeric RES complex is organized around the Snu17p protein that binds to both the Bud13p and Pml1p subunits. Snu17p exhibits an RRM domain that resembles a U2AF homology motif (UHM) and Bud13p harbors a Trp residue reminiscent of an UHM-ligand motif (ULM). It has therefore been proposed that the interaction between Snu17p and Bud13p resembles canonical UHM-ULM complexes. Here, we have used biochemical and NMR structural analysis to characterize the structure of the yeast Snu17p-Bud13p complex. Unlike known UHMs that sequester the Trp residue of the ULM ligand in a hydrophobic pocket, Snu17p and Bud13p utilize a large interaction surface formed around the two helices of the Snu17p domain. In total 18 residues of the Bud13p ligand wrap around the Snu17p helical surface in an U-turn-like arrangement. The invariant Trp232 in Bud13p is located in the center of the turn, and contacts surface residues of Snu17p. The structural data are supported by mutational analysis and indicate that Snu17p provides an extended binding surface with Bud13p that is notably distinct from canonical UHM-ULM interactions. Our data highlight structural diversity in RRM-protein interactions, analogous to the one seen for nucleic acid interactions.

Crystal Structure of PAV1-137: A Protein from the Virus PAV1 That Infects Pyrococcus abyssi

Pyrococcus abyssi virus 1 (PAV1) was the first virus particle infecting a hyperthermophilic Euryarchaeota (Pyrococcus abyssi strain GE23) that has been isolated and characterized. It is lemon shaped and is decorated with a short fibered tail. PAV1 morphologically resembles the fusiform members of the family Fuselloviridae or the genus Salterprovirus. The 18 kb dsDNA genome of PAV1 contains 25 predicted genes, most of them of unknown function. To help assigning functions to these proteins, we have initiated structural studies of the PAV1 proteome. We determined the crystal structure of a putative protein of 137 residues (PAV1-137) at a resolution of 2.2 Å. The protein forms dimers both in solution and in the crystal. The fold of PAV1-137 is a four-α-helical bundle analogous to those found in some eukaryotic adhesion proteins such as focal adhesion kinase, suggesting that PAV1-137 is involved in protein-protein interactions.