The structure of Yersinia pestis V-antigen, an essential virulence factor and mediator of immunity against plague.

The LcrV protein (V-antigen) is a multifunctional virulence factor in Yersinia pestis, the causative agent of plague. LcrV regulates the translocation of cytotoxic effector proteins from the bacterium into the cytosol of mammalian cells via a type III secretion system, possesses antihost activities of its own, and is also an active and passive mediator of resistance to disease. Although a crystal structure of this protein has been actively sought for better understanding of its role in pathogenesis, the wild-type LcrV was found to be recalcitrant to crystallization.

Similar modes of polypeptide recognition by export chaperones in flagellar biosynthesis and type III secretion.

Assembly of the bacterial flagellum and type III secretion in pathogenic bacteria require cytosolic export chaperones that interact with mobile components to facilitate their secretion. Although their amino acid sequences are not conserved, the structures of several type III secretion chaperones revealed striking similarities between their folds and modes of substrate recognition. Here, we report the first crystallographic structure of a flagellar export chaperone, Aquifex aeolicus FliS.

Differential effects of supplementary affinity tags on the solubility of MBP fusion proteins.

It is difficult to imagine any strategy for high-throughput protein expression and purification that does not involve genetically engineered affinity tags. Because of its ability to enhance the solubility and promote the proper folding of its fusion partners, Escherichia coli maltose-binding protein (MBP) is a particularly useful affinity tag. However, not all MBP fusion proteins bind efficiently to amylose resin, and even when they do it is usually not possible to obtain a sample of adequate purity after a single affinity step.

Characterization of four orthologs of stringent starvation protein A.

Orthologous proteins can be beneficial for X-ray crystallographic studies when a protein from an organism of choice fails to crystallize or the crystals are not suitable for structure determination. Their amino-acid sequences should be similar enough that they will share the same fold, but different enough so that they may crystallize under alternative conditions and diffract to higher resolution. This multi-species approach was employed to obtain diffraction-quality crystals of the RNA polymerase (RNAP) associated stringent starvation protein A (SspA).

Maltodextrin-binding proteins from diverse bacteria and archaea are potent solubility enhancers.

Escherichia coli maltose-binding protein (MBP) is frequently used as an affinity tag to facilitate the purification of recombinant proteins. An important additional attribute of MBP is its remarkable ability to enhance the solubility of its fusion partners. MBPs are present in a wide variety of microorganisms including both mesophilic and thermophilic bacteria and archaea.

A spring-loaded state of NusG in its functional cycle is suggested by X-ray crystallography and supported by site-directed mutants.

Transcription factor NusG is present in all prokaryotes, and orthologous proteins have also been identified in yeast and humans. NusG contains a 27-residue KOW motif, found in ribosomal protein L24 where it interacts with rRNA. NusG in Escherichia coli (EcNusG) is an essential protein and functions as a regulator of Rho-dependent transcription termination, phage lambda N and rRNA transcription antitermination, and phage HK022 Nun termination.

Crystallization and preliminary X-ray diffraction studies of NusG, a protein shared by the transcription and translation machines.

N-utilization factor G (NusG) from Aquifex aeolicus (Aa) was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapor-diffusion technique. The drops consisted of 2.5 microl protein solution (approximately 30 mg ml(-1) in 20 mM Tris-HCl pH 8.

Three-dimensional structure of the type III secretion chaperone SycE from Yersinia pestis.

Many bacterial pathogens utilize a type III (contact-dependent) secretion system to inject cytotoxic effector proteins directly into host cells. This ingenious mechanism, designed for both bacterial offense and defense, has been studied most extensively in Yersinia spp. To be exported efficiently, at least three of the effectors (YopE, YopH and YopT) and several other proteins that transit the type III secretion pathway in Yersinia (YopN, YopD and YopB) must first form transient complexes with cognate-specific Yop chaperone (Syc) proteins.

Processive degradation of nascent polypeptides, triggered by tandem AGA codons, limits the accumulation of recombinant tobacco etch virus protease in Escherichia coli BL21(DE3).

Due to its high degree of sequence specificity, the catalytic domain of the nuclear inclusion protease from tobacco etch virus (TEV protease) is a useful reagent for cleaving genetically engineered fusion proteins. However, the overproduction of TEV protease in Escherichia coli has been hampered in the past by low yield and poor solubility. Here we demonstrate that the low yield can be attributed to the presence of arginine codons in the TEV protease coding sequence that are rarely used in E.

Crystal structure of the Yersinia pestis GTPase activator YopE.

Yersinia pestis, the causative agent of bubonic plague, evades the immune response of the infected organism by using a type III (contact-dependent) secretion system to deliver effector proteins into the cytosol of mammalian cells, where they interfere with signaling pathways that regulate inflammation and cytoskeleton dynamics. The cytotoxic effector YopE functions as a potent GTPase-activating protein (GAP) for Rho family GTP-binding proteins, including RhoA, Rac1, and Cdc42. Down-regulation of these molecular switches results in the loss of cell motility and inhibition of phagocytosis, enabling Y.