TALL-1͞Blys͞BAFF is a member of the tumor necrosis factor (TNF) ligand superfamily that is functionally involved in B cell proliferation. Here, we describe B cell hyperplasia and autoimmune lupuslike changes in transgenic mice expressing TALL-1 under the control of a ␤-actin promoter. The TALL-1 transgenic mice showed severe enlargement of spleen, lymph nodes, and Peyer's patches because of an increased number of B220؉ cells. The transgenic mice also had hypergammaglobulinemia contributed by elevations of serum IgM, IgG, IgA, and IgE. In addition, a phenotype similar to autoimmune lupus-like disease was also seen in TALL-1 transgenic mice, characterized by the presence of autoantibodies to nuclear antigens and immune complex deposits in the kidney. Prolonged survival and hyperactivity of transgenic B cells may contribute to the autoimmune lupus-like phenotype in these animals. Our studies further confirm TALL-1 as a stimulator of B cells that affect Ig production. Thus, TALL-1 may be a primary mediator in B cellassociated autoimmune diseases.TNF ligand family ͉ B cell stimulation

The essential cell division protein, FtsZ, from Mycobacterium tuberculosis has been expressed in Escherichia coli and purified. The recombinant protein has GTPase activity typical of tubulin and other FtsZs. FtsZ polymerization was studied using 90°light scattering. The mycobacterial protein reaches maximum polymerization much more slowly (ϳ10 min) than E. coli FtsZ. Depolymerization also occurs slowly, taking 1 h or longer under most conditions. Polymerization requires both Mg 2؉ Bacteria have a variety of genes that are critical for cell division, among them the fts genes (reviewed in references 20, 24, and 26). Cell division occurs at the site of formation of the contractile Z ring, which is composed of a polymer of FtsZ. FtsZ, a 40-kDa protein, is ubiquitous in eubacteria and archaea. Although it has only weak sequence homology to mammalian tubulin, it does contain the tubulin signature motif GGGTGS/TG (7, 9), which is believed to be necessary for the GTPase activity of tubulin. FtsZ polymerizes to form the Z ring in a GTP-dependent manner, analogous to the polymerization of tubulin to form microtubules (4,6,10,21,32). The three-dimensional structures of Methanococcus jannaschii FtsZ and ␣-and ␤-tubulin are quite similar (16,17) and reveal that FtsZ and tubulin form a unique family of GTPases (24).Although the polymerization of tubulin has been studied extensively, understanding FtsZ polymerization has been hampered until recently by the lack of a rapid, easy assay. To date the primary method of examining FtsZ polymerization has relied on centrifugation of the reaction mixture followed by an examination of the pellet or by electron microscopy. Most of the published work has dealt with FtsZ from Escherichia coli (1,6,20). Recently, Mukherjee and Lutkenhaus (22) introduced a light-scattering assay that uses a fluorometer to monitor polymer formation and dissolution. We have used this method to extend our understanding of FtsZ by examining the dynamics of Mycobacterium tuberculosis FtsZ polymerization in vitro. M. tuberculosis FtsZ polymerization is similar to that of E. coli FtsZ in many respects. However, there are some significant differences between the two, with M. tuberculosis FtsZ showing some characteristics more reminiscent of its homolog tubulin than the E. coli protein. This work represents the first study of M. tuberculosis FtsZ, a critical cell division protein for a pathogenic organism of worldwide medical importance. MATERIALS AND METHODSPurification of FtsZ. The M. tuberculosis FtsZ coding sequence was subcloned into the NcoI site of pET15b (Novagen). The resulting plasmid, pJD168, was used to transform E. coli BL21(DE3)/pLysS. Cells were incubated at 32°C in Luria-Bertani (LB) media containing 0.4% glucose for 1 h. Five hundred microliters of transformed cells was added to 250 ml of fresh LB medium containing 0.4% glucose, 100 g of ampicillin/ml, and 34 g of chloramphenicol/ml and incubated overnight at 32°C. The cells were pelleted by centrifugation at room temperature. They were then resusp...

The crystal structure of the Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-xanthosine 5'-monophosphate (XMP)-pyrophosphate-Mg(2+) ternary complex has been determined at 1. 60 A resolution. This biproduct, post-transition state structure is of a T. gondii HGPRT mutant (Asp150Ala or D150A). The D150A mutant has reduced activity (k(cat) lower by 11-, 296-, and 8.6-fold for hypoxanthine, guanine, and xanthine, respectively) compared to wild-type T. gondii HGPRT. The Michaelis constants for purine bases are altered only slightly, whereas those for alpha-D-5-phosphoribosyl 1-pyrophosphate (PRPP) are lower by approximately 6.5-fold. The ternary complex crystallizes in space group C222(1) (a = 55.21 A, b = 112.25 A, and c = 144.28 A) with two subunits in the asymmetric unit; the HGPRT tetramer is completed by the application of 2-fold crystallographic symmetry. All active sites contain XMP ¿bound in a fashion similar to that of the guanosine 5'-monophosphate (GMP) and inosine 5'-monophosphate (IMP) complexes reported in the preceding article [Héroux, A., et al. (1999) Biochemistry 38, 14485-14494]¿, pyrophosphate, and two Mg(2+) ions. Each Mg(2+) ion is octahedrally coordinated by two terminal pyrophosphate oxygen atoms and several ordered water molecules. This structure shows how HGPRT uses two Mg(2+) ions to orient and activate the pyrophosphate moiety of PRPP for attack by a purine base, and why mutation in humans of the residue corresponding to Asp206, the only HGPRT amino acid that directly contacts the Mg(2+) ions, causes Lesch-Nyhan syndrome (HGPRT(Kinston), D193N). The Leu78-Lys79 peptide bond in the active site adopts the cis configuration, which it must to bind PRPP or pyrophosphate. The contribution of cis-trans isomerization of this peptide bond to the energetics of substrate binding and product release is discussed. A comprehensive description of the HGPRT reaction mechanism is also proposed.

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The crystal structures of the guanosine 5'-monophosphate (GMP) and inosine 5'-monophosphate (IMP) complexes of Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase (HGPRT) have been determined at 1.65 and 1.90 A resolution. These complexes, which crystallize in space groups P2(1) (a = 65.45 A, b = 90.84 A, c = 80. 26 A, and beta = 92.53 degrees ) and P2(1)2(1)2(1) (a = 84.54 A, b = 102.44 A, and c = 108.83 A), each comprise a tetramer in the crystallographic asymmetric unit. All active sites in the tetramers are fully occupied by the nucleotide. Comparison of these structures with that of the xanthosine 5'-monophosphate (XMP)-pyrophosphate-Mg(2+) ternary complex reported in the following article [Héroux, A., et al. (1999) Biochemistry 38, 14495-14506] shows how T. gondii HGPRT is able to recognize guanine, hypoxanthine, and xanthine as substrates, and suggests why the human enzyme cannot use xanthine efficiently. Comparison with the apoenzyme reveals the structural changes that occur upon binding of purines and ribose 5'-phosphate to HGPRT. Two structural features important to the HGPRT mechanism, a previously unrecognized active site loop (loop III', residues 180-184) and an active site peptide bond (Leu78-Lys79) that adopts both the cis and the trans configurations, are presented.

Three types of substrate deformation, chief among them an unexpected C2'-endo pucker adopted by the PRPP ribose ring, raise the energy of the ground state. A cation-pi interaction between Tyr-118 and the developing oxocarbenium ion in the ribose ring helps to stabilize the transition state. Enforced substrate propinquity coupled with optimal reactive geometry for both the substrates and the active site residues with which they interact contributes to catalysis as well.

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