DNA ligases catalyze the crucial step of joining the breaks in duplex DNA during DNA replication, repair and recombination, utilizing either ATP or NAD ⍣ as a cofactor. Despite the difference in cofactor specificity and limited overall sequence similarity, the two classes of DNA ligase share basically the same catalytic mechanism. In this study, the crystal structure of an NAD ⍣ -dependent DNA ligase from Thermus filiformis, a 667 residue multidomain protein, has been determined by the multiwavelength anomalous diffraction (MAD) method. It reveals highly modular architecture and a unique circular arrangement of its four distinct domains. It also provides clues for protein flexibility and DNA-binding sites. A model for the multidomain ligase action involving large conformational changes is proposed.

Cyclic diguanylate (c-di-GMP) is a ubiquitous second messenger regulating diverse cellular functions including motility, biofilm formation, cell cycle progression and virulence in bacteria. In the cell, degradation of c-di-GMP is catalyzed by highly specific EAL domain phosphodiesterases whose catalytic mechanism is still unclear. Here, we purified 13 EAL domain proteins from various organisms and demonstrated that their catalytic activity is associated with the presence of 10 conserved EAL domain residues. The crystal structure of the TDB1265 EAL domain was determined in a free state (1.8 Å) and in complex with c-di-GMP (2.35 Å) and unveiled the role of the conserved residues in substrate binding and catalysis. The structure revealed the presence of two metal ions directly coordinated by six conserved residues, two oxygens of the c-di-GMP phosphate, and potential catalytic water molecule. Our results support a two-metal-ion catalytic mechanism of c-di-GMP hydrolysis by EAL domain phosphodiesterases.

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We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.

The TonB-dependent complex of Gram-negative bacteria couples the inner membrane proton motive force to the active transport of iron⅐siderophore and vitamin B 12 across the outer membrane. The structural basis of that process has not been described so far in full detail. The crystal structure of the C-terminal domain of TonB from Escherichia coli has now been solved by multiwavelength anomalous diffraction and refined at 1.55-Å resolution, providing the first evidence that this region of TonB (residues 164 -239) dimerizes. Moreover, the structure shows a novel architecture that has no structural homologs among any known proteins. The dimer of the C-terminal domain of TonB is cylinder-shaped with a length of 65 Å and a diameter of 25 Å. Each monomer contains three ␤ strands and a single ␣ helix. The two monomers are intertwined with each other, and all six ␤-strands of the dimer make a large antiparallel ␤-sheet. We propose a plausible model of binding of TonB to FhuA and FepA, two TonB-dependent outer-membrane receptors.The outer membrane (OM 1 ) of Gram-negative bacteria constitutes a permeability barrier, protecting the cell against a variety of toxic agents. The lipopolysaccharides located in the outer leaflet of the OM confer to the bacteria a polar and negatively charged surface, restricting the cellular uptake of toxic organic molecules and detergents such as bile salts, the detergents in the gut. However, although the OM is an effective protective barrier against harmful environmental components, it also represents an additional obstacle for the uptake of nutrients, which can be circumvented in three ways. While small hydrophilic nutrients (Ͻ600 Da) enter the periplasm by simple diffusion through porins in a non-selective manner (1), larger molecules are taken up by pores with an internal binding site for the ligand (such as LamB) in a stereospecific and selective manner (2) and can subsequently enter the cytoplasm by a variety of transporters located in the inner membrane (3). A few nutrients, notably iron and vitamin B 12 , need to be taken up into the periplasm against their concentration gradients.For this purpose, a complex consisting of TonB, ExbB, and ExbD couples the inner membrane proton motive force (pmf) to the active transport of iron siderophores and vitamin B 12 across the OM through specialized porins. Recently, crystal structures were solved for two TonB-dependent receptors, . Like all other known porins, they are ␤-barrels, but unlike the other porin structures they have much larger interiors, which are almost completely obscured by a protein domain sitting inside the barrel (termed the "cork" or "hatch region"), which is encoded within the N-terminal segment of either protein.Iron uptake into bacteria is initiated by the binding of the iron⅐siderophore complex to the high affinity OM receptor. The dissociation constant is around 100 -200 nM (7,8). An electron spin resonance study (9), later rationalized by three-dimensional structural models (4 -6), has shown that this event triggers conf...

Glutaminases belong to the large superfamily of serine-dependent beta-lactamases and penicillin-binding proteins, and they catalyze the hydrolytic deamidation of L-glutamine to L-glutamate. In this work, we purified and biochemically characterized four predicted glutaminases from Escherichia coli (YbaS and YneH) and Bacillus subtilis (YlaM and YbgJ). The proteins demonstrated strict specificity to L-glutamine and did not hydrolyze D-glutamine or L-asparagine. In each organism, one glutaminase showed higher affinity to glutamine ( E. coli YbaS and B. subtilis YlaM; K m 7.3 and 7.6 mM, respectively) than the second glutaminase ( E. coli YneH and B. subtilis YbgJ; K m 27.6 and 30.6 mM, respectively). The crystal structures of the E. coli YbaS and the B. subtilis YbgJ revealed the presence of a classical beta-lactamase-like fold and conservation of several key catalytic residues of beta-lactamases (Ser74, Lys77, Asn126, Lys268, and Ser269 in YbgJ). Alanine replacement mutagenesis demonstrated that most of the conserved residues located in the putative glutaminase catalytic site are essential for activity. The crystal structure of the YbgJ complex with the glutaminase inhibitor 6-diazo-5-oxo- l-norleucine revealed the presence of a covalent bond between the inhibitor and the hydroxyl oxygen of Ser74, providing evidence that Ser74 is the primary catalytic nucleophile and that the glutaminase reaction proceeds through formation of an enzyme-glutamyl intermediate. Growth experiments with the E. coli glutaminase deletion strains revealed that YneH is involved in the assimilation of l-glutamine as a sole source of carbon and nitrogen and suggested that both glutaminases (YbaS and YneH) also contribute to acid resistance in E. coli.

Legionella pneumophila translocates the largest known arsenal of over 330 pathogenic factors, called "effectors," into host cells during infection, enabling L. pneumophila to establish a replicative niche inside diverse amebas and human macrophages. Here, we reveal that the L. pneumophila effectors MavC (Lpg2147) and MvcA (Lpg2148) are structural homologs of cycle inhibiting factor (Cif) effectors and that the adjacent gene, lpg2149, produces a protein that directly inhibits their activity. In contrast to canonical Cifs, both MavC and MvcA contain an insertion domain and deamidate the residue Gln40 of ubiquitin but not Gln40 of NEDD8. MavC and MvcA are functionally diverse, with only MavC interacting with the human E2-conjugating enzyme UBE2N (Ubc13). MavC deamidates the UBE2N∼Ub conjugate, disrupting Lys63 ubiquitination and dampening NF-κB signaling. Combined, our data reveal a molecular mechanism of host manipulation by pathogenic bacteria and highlight the complex regulatory mechanisms integral to L. pneumophila's pathogenic strategy.

L-Proline is an amino acid that plays an important role in proteins uniquely contributing to protein folding, structure, and stability, and this amino acid serves as a sequence-recognition motif. Proline biosynthesis can occur via two pathways, one from glutamate and the other from arginine. In both pathways, the last step of biosynthesis, the conversion of Δ 1 -pyrroline-5-carboxylate (P5C) to Lproline, is catalyzed by Δ 1 -pyrroline-5-carboxylate reductase (P5CR) using NAD(P)H as a cofactor. We have determined the first crystal structure of P5CR from two human pathogens, Neisseria meningitides and Streptococcus pyogenes, at 2.0Å and 2.15Å resolution, respectively. The catalytic unit of P5CR is a dimer composed of two domains, but the biological unit seems to be speciesspecific. The N-terminal domain of P5CR is an α/β/α sandwich, a Rossmann fold. The C-terminal dimerization domain is rich in α-helices and shows domain swapping. Comparison of the native structure of P5CR to structures complexed with L-proline and NADP + in two quite different primary sequence backgrounds provides unique information about key functional features: the active site and the catalytic mechanism. The inhibitory L-proline has been observed in the crystal structure.