Crystallographic and modeling studies of RNase III suggest a mechanism for double-stranded RNA cleavage.

BACKGROUND: Aquifex aeolicus Ribonuclease III (Aa-RNase III) belongs to the family of Mg(2+)-dependent endonucleases that show specificity for double-stranded RNA (dsRNA). RNase III is conserved in all known bacteria and eukaryotes and has 1-2 copies of a 9-residue consensus sequence, known as the RNase III signature motif. The bacterial RNase III proteins are the simplest, consisting of two domains: an N-terminal endonuclease domain, followed by a double-stranded RNA binding domain (dsRBD). The three-dimensional structure of the dsRBD in Escherichia coli RNase III has been elucidated; no structural information is available for the endonuclease domain of any RNase III. RESULTS: We present the crystal structures of the Aa-RNase III endonuclease domain in its ligand-free form and in complex with Mn(2+). The structures reveal a novel protein fold and suggest a mechanism for dsRNA cleavage. On the basis of structural, genetic, and biological data, we have constructed a hypothetical model of Aa-RNase III in complex with dsRNA and Mg(2+) ion, which provides the first glimpse of RNase III in action. CONCLUSIONS: The functional Aa-RNase III dimer is formed via mainly hydrophobic interactions, including a "ball-and-socket" junction that ensures accurate alignment of the two monomers. The fold of the polypeptide chain and its dimerization create a valley with two compound active centers at each end of the valley. The valley can accommodate a dsRNA substrate. Mn(2+) binding has significant impact on crystal packing, intermolecular interactions, thermal stability, and the formation of two RNA-cutting sites within each compound active center.

Unusual molecular architecture of the Yersinia pestis cytotoxin YopM: a leucine-rich repeat protein with the shortest repeating unit.

Many Gram-negative bacterial pathogens employ a contact-dependent (type III) secretion system to deliver effector proteins into the cytosol of animal or plant cells. Collectively, these effectors enable the bacteria to evade the immune response of the infected organism by modulating host-cell functions. YopM, a member of the leucine-rich repeat protein superfamily, is an effector produced by the bubonic plague bacterium, Yersinia pestis, that is essential for virulence. Here, we report crystal structures of YopM at 2.4 and 2.1 A resolution. Among all leucine-rich repeat family members whose atomic coordinates have been reported, the repeating unit of YopM has the least canonical secondary structure. In both crystals, four YopM monomers form a hollow cylinder with an inner diameter of 35 A. The domain that targets YopM for translocation into eukaryotic cells adopts a well-ordered, alpha-helical conformation that packs tightly against the proximal leucine-rich repeat module. A similar alpha-helical domain can be identified in virulence-associated leucine-rich repeat proteins produced by Salmonella typhimurium and Shigella flexneri, and in the conceptual translation products of several open reading frames in Y. pestis.

Structure of the N-terminal domain of Yersinia pestis YopH at 2.0 A resolution.

Yersinia pestis, the causative agent of bubonic plague, injects effector proteins into the cytosol of mammalian cells that enable the bacterium to evade the immune response of the infected organism by interfering with eukaryotic signal transduction pathways. YopH is a modular effector composed of a C-terminal protein tyrosine phosphatase (PTPase) domain and a multifunctional N-terminal domain that not only orchestrates the secretion and translocation of YopH into eukaryotic cells but also binds tyrosine-phosphorylated target proteins to mediate substrate recognition. The crystal structure of the N-terminal domain of YopH (YopH(N); residues 1-130) has been determined at 2.0 A resolution. The amino-acid sequences that target YopH for secretion from the bacterium and translocation into eukaryotic cells form integral parts of this compactly folded domain. The structure of YopH(N) bears no resemblance to eukaryotic phosphotyrosine-binding domains, nor is it reminiscent of any known fold. Residues that have been implicated in phosphotyrosine-dependent protein binding are clustered together on one face of YopH(N), but the structure does not suggest a mechanism for protein-phosphotyrosine recognition.

Structural basis for oligosaccharide recognition by Pyrococcus furiosus maltodextrin-binding protein.

A maltodextrin-binding protein from Pyrococcus furiosus (PfuMBP) has been overproduced in Escherichia coli, purified, and crystallized. The crystal structure of the protein bound to an oligosaccharide ligand was determined to 1.85 A resolution. The fold of PfuMBP is very similar to that of the orthologous MBP from E. coli (EcoMBP), despite the moderate level of sequence identity between the two proteins (27 % identity, 46 % similarity). PfuMBP is extremely resistant to heat and chemical denaturation, which may be attributed to a number of factors, such as a tightly packed hydrophobic core, clusters of isoleucine residues, salt-bridges, and the presence of proline residues in key positions. Surprisingly, an attempt to crystallize the complex of PfuMBP with maltose resulted in a structure that contained maltotriose in the ligand-binding site. The structure of the complex suggests that there is a considerable energy gain upon binding of maltotriose in comparison to maltose. Moreover, isothermal titration calorimetry experiments demonstrated that the binding of maltotriose to the protein is exothermic and tight, whereas no thermal effect was observed upon addition of maltose at three temperatures. Therefore, PfuMBP evidently is designed to bind oligosaccharides composed of three or more glucopyranose units.

Overproduction, purification, crystallization and preliminary X-ray diffraction analysis of YopM, an essential virulence factor extruded by the plague bacterium Yersinia pestis.

A recombinant form of Yersinia pestis YopM with a C-terminal polyhistidine affinity tag has been overproduced in Escherichia coli, purified to homogeneity and crystallized using the hanging-drop vapor-diffusion technique. Several different crystal forms were obtained. The most suitable crystals for X-ray diffraction belonged to space groups P4(2)22 (unit-cell parameters a = 109.36, b = 109.36, c = 101.50 A) and C222(1) (unit-cell parameters a = 71.73, b = 121. 85, c = 189.79 A). With a synchrotron-radiation source, these crystals diffracted to 2.4 and 1.9 A resolution, respectively.