Complex Formation between Mur Enzymes from Streptococcus pneumoniae.

Peptidoglycan is one of the major components of the bacterial cell wall, being responsible for shape and stability. Due to its essential nature, its biosynthetic pathway is the target for major antibiotics, and proteins involved in its biosynthesis continue to be targeted for inhibitor studies. The biosynthesis of its major building block, Lipid II, is initiated in the bacterial cytoplasm with the sequential reactions catalyzed by Mur enzymes, which have been suggested to form a multiprotein complex to facilitate shuttling of the building blocks toward the inner membrane. In this work, we purified MurC, MurD, MurE, MurF, and MurG from the human pathogen Streptococcus pneumoniae and characterized their interactions using chemical cross-linking, mass spectrometry, analytical ultracentrifugation, and microscale thermophoresis. Mur ligases interact strongly as binary complexes, with interaction regions mapping mostly to loop regions. Interestingly, MurC, MurD, and MurE display 10-fold higher affinity for each other than for MurF and MurG, suggesting that Mur ligases that catalyze the initial reactions in the peptidoglycan biosynthesis pathway could form a subcomplex that could be important to facilitate Lipid II biosynthesis. The interface between Mur proteins could represent a yet unexplored target for new inhibitor studies that could lead to the development of novel antimicrobials.

Assembly of an atypical α-macroglobulin complex from Pseudomonas aeruginosa.

Alpha-2-macroglobulins (A2Ms) are large spectrum protease inhibitors that are major components of the eukaryotic immune system. Pathogenic and colonizing bacteria, such as the opportunistic pathogen Pseudomonas aeruginosa, also carry structural homologs of eukaryotic A2Ms. Two types of bacterial A2Ms have been identified: Type I, much like the eukaryotic form, displays a conserved thioester that is essential for protease targeting, and Type II, which lacks the thioester and to date has been poorly studied despite its ubiquitous presence in Gram-negatives. Here we show that MagD, the Type II A2M from P. aeruginosa that is expressed within the six-gene mag operon, specifically traps a target protease despite the absence of the thioester motif, comforting its role in protease inhibition. In addition, analytical ultracentrifugation and small angle scattering show that MagD forms higher order complexes with proteins expressed in the same operon (MagA, MagB, and MagF), with MagB playing the key stabilization role. A P. aeruginosa strain lacking magB cannot stably maintain MagD in the bacterial periplasm, engendering complex disruption. This suggests a regulated mechanism of Mag complex formation and stabilization that is potentially common to numerous Gram-negative organisms, and that plays a role in periplasm protection from proteases during infection or colonization.

The Trypanosoma cruzi virulence factor oligopeptidase B (OPBTc) assembles into an active and stable dimer.

Oligopeptidase B, a processing enzyme of the prolyl oligopeptidase family, is considered as an important virulence factor in trypanosomiasis. Trypanosoma cruzi oligopeptidase B (OPBTc) is involved in host cell invasion by generating a Ca(2+)-agonist necessary for recruitment and fusion of host lysosomes at the site of parasite attachment. The underlying mechanism remains unknown and further structural and functional characterization of OPBTc may help clarify its physiological function and lead to the development of new therapeutic molecules to treat Chagas disease. In the present work, size exclusion chromatography and analytical ultracentrifugation experiments demonstrate that OPBTc is a dimer in solution, an association salt and pH-resistant and independent of intermolecular disulfide bonds. The enzyme retains its dimeric structure and is fully active up to 42°C. OPBTc is inactivated and its tertiary, but not secondary, structure is disrupted at higher temperatures, as monitored by circular dichroism and fluorescence spectroscopy. It has a highly stable secondary structure over a broad range of pH, undergoes subtle tertiary structure changes at low pH and is less stable under moderate ionic strength conditions. These results bring new insights into the structural properties of OPBTc, contributing to future studies on the rational design of OPBTc inhibitors as a promising strategy for Chagas disease chemotherapy.

The major leucyl aminopeptidase of Trypanosoma cruzi (LAPTc) assembles into a homohexamer and belongs to the M17 family of metallopeptidases.

BACKGROUND: Pathogens depend on peptidase activities to accomplish many physiological processes, including interaction with their hosts, highlighting parasitic peptidases as potential drug targets. In this study, a major leucyl aminopeptidolytic activity was identified in Trypanosoma cruzi, the aetiological agent of Chagas disease. RESULTS: The enzyme was isolated from epimastigote forms of the parasite by a two-step chromatographic procedure and associated with a single 330-kDa homohexameric protein as determined by sedimentation velocity and light scattering experiments. Peptide mass fingerprinting identified the enzyme as the predicted T. cruzi aminopeptidase EAN97960. Molecular and enzymatic analysis indicated that this leucyl aminopeptidase of T. cruzi (LAPTc) belongs to the peptidase family M17 or leucyl aminopeptidase family. LAPTc has a strong dependence on neutral pH, is mesophilic and retains its oligomeric form up to 80°C. Conversely, its recombinant form is thermophilic and requires alkaline pH. CONCLUSIONS: LAPTc is a 330-kDa homohexameric metalloaminopeptidase expressed by all T. cruzi forms and mediates the major parasite leucyl aminopeptidolytic activity. Since biosynthetic pathways for essential amino acids, including leucine, are lacking in T. cruzi, LAPTc could have a function in nutritional supply.

Structure and interactions of fish type III antifreeze protein in solution.

It has been suggested that above a critical protein concentration, fish Type III antifreeze protein (AFP III) self-assembles to form micelle-like structures that may play a key role in antifreeze activity. To understand the complex activity of AFP III, a comprehensive description of its association state and structural organization in solution is necessary. We used analytical ultracentrifugation, analytical size-exclusion chromatography, and dynamic light scattering to characterize the interactions and homogeneity of AFP III in solution. Small-angle neutron scattering was used to determine the low-resolution structure in solution. Our results clearly show that at concentrations up to 20 mg mL(-1) and at temperatures of 20 degrees C, 6 degrees C, and 4 degrees C, AFP III is monomeric in solution and adopts a structure compatible with that determined by crystallography. Surface tension measurements show a propensity of AFP III to localize at the air/water interface, but this surface activity is not correlated with any aggregation in the bulk. These results support the hypothesis that each AFP III molecule acts independently of the others, and that specific intermolecular interactions between monomers are not required for binding to ice. The lack of attractive interactions between monomers may be functionally important, allowing for more efficient binding and covering of the ice surface.