Alejandro Buschiazzo

The crystal structure and mode of action of trans-sialidase, a key enzyme in Trypanosoma cruzi pathogenesis

Trans-sialidases (TS) are GPI-anchored surface enzymes expressed in specific developmental stages of trypanosome parasites like Trypanosoma cruzi, the etiologic agent of Chagas disease, and T. brucei, the causative agent of sleeping sickness. TS catalyzes the transfer of sialic acid residues from host to parasite glycoconjugates through a transglycosidase reaction that appears to be critical for T. cruzi survival and cell invasion capability. We report here the structure of the T. cruzi trans-sialidase, alone and in complex with sugar ligands. Sialic acid binding is shown to trigger a conformational switch that modulates the affinity for the acceptor substrate and concomitantly creates the conditions for efficient transglycosylation. The structure provides a framework for the structure-based design of novel inhibitors with potential therapeutic applications.

Structural basis of sialyltransferase activity in trypanosomal sialidases

The intracellular parasite Trypanosoma cruzi, the etiological agent of Chagas disease, sheds a developmentally regulated surface trans‐sialidase, which is involved in key aspects of parasite–host cell interactions. Although it shares a common active site architecture with bacterial neuraminidases, the T.cruzi enzyme behaves as a highly efficient sialyltransferase. Here we report the crystal structure of the closely related Trypanosoma rangeli sialidase and its complex with inhibitor. The enzyme folds into two distinct domains: a catalytic β‐propeller fold tightly associated with a lectin‐like domain. Comparison with the modeled structure of T.cruzi trans‐sialidase and mutagenesis experiments allowed the identification of amino acid substitutions within the active site cleft that modulate sialyltransferase activity and suggest the presence of a distinct binding site for the acceptor carbohydrate. The structures of the Trypanosoma enzymes illustrate how a glycosidase scaffold can achieve efficient glycosyltransferase activity and provide a framework for structure‐based drug design.

Crystal structure of glycogen synthase: homologous enzymes catalyze glycogen synthesis and degradation.

Glycogen and starch are the major readily accessible energy storage compounds in nearly all living organisms. Glycogen is a very large branched glucose homopolymer containing about 90% α‐1,4‐glucosidic linkages and 10% α‐1,6 linkages. Its synthesis and degradation constitute central pathways in the metabolism of living cells regulating a global carbon/energy buffer compartment. Glycogen biosynthesis involves the action of several enzymes among which glycogen synthase catalyzes the synthesis of the α‐1,4‐glucose backbone. We now report the first crystal structure of glycogen synthase in the presence and absence of adenosine diphosphate. The overall fold and the active site architecture of the protein are remarkably similar to those of glycogen phosphorylase, indicating a common catalytic mechanism and comparable substrate‐binding properties. In contrast to glycogen phosphorylase, glycogen synthase has a much wider catalytic cleft, which is predicted to undergo an important interdomain ‘closure’ movement during the catalytic cycle. The structures also provide useful hints to shed light on the allosteric regulation mechanisms of yeast/mammalian glycogen synthases.

Identification of the gene(s) coding for the trans-sialidase of Trypanosoma cruzi.

The gene(s) encoding the Trypanosoma cruzi shed‐acute‐phase‐antigen (SAPA) has a 5′ end encoding a region containing two totally and two partially conserved Ser‐X‐Asp‐X‐Gly‐X‐Thr‐Trp motifs which are present in bacterial neuraminidases, and a 3′ end encoding tandemly repeated units of 12 amino acids. It is now reported that 54–87% of the total neuraminidase activity present in the parasite could be immunoprecipitated with polyclonal or monoclonal antibodies against the repeated amino acid units of SAPA. These immunoprecipitates also had greater than 80% of the trans‐sialidase activity of the parasite. SAPA used sialyllactose, fetuin and 4‐methylumbelliferyl‐sialic acid as substrate donors. In the presence of a suitable acceptor molecule (lactose) the sialic acid residues were transferred to the disaccharide, whereas in the absence of acceptors the residues were transferred to water. If relatively inefficient acceptors (maltose or cellobiose) were added to the incubation mixtures, the sialic acid units were transferred both to the disaccharides and to water. It is concluded that a major T. cruzi antigen has both the trans‐sialidase and the neuraminidase activities of the parasite. Both activities are probably located on the N‐terminus of SAPA since antibodies directed against the C‐terminus, which contains the repeated amino acid units, do not affect the enzymatic activities.

Active nuclear import and cytoplasmic retention of activation-induced deaminase

The enzyme activation-induced deaminase (AID) triggers antibody diversification in B cells by catalyzing deamination and consequently mutation of immunoglobulin genes. To minimize off-target deamination, AID is restrained by several regulatory mechanisms including nuclear exclusion, thought to be mediated exclusively by active nuclear export. Here we identify two other mechanisms involved in controlling AID subcellular localization. AID is unable to passively diffuse into the nucleus, despite its small size, and its nuclear entry requires active import mediated by a conformational nuclear localization signal. We also identify in its C terminus a determinant for AID cytoplasmic retention, which hampers diffusion to the nucleus, competes with nuclear import and is crucial for maintaining the predominantly cytoplasmic localization of AID in steady-state conditions. Blocking nuclear import alters the balance between these processes in favor of cytoplasmic retention, resulting in reduced isotype class switching.

Structural plasticity and catalysis regulation of a thermosensor histidine kinase

Temperature sensing is essential for the survival of living cells. A major challenge is to understand how a biological thermometer processes thermal information to optimize cellular functions. Using structural and biochemical approaches, we show that the thermosensitive histidine kinase, DesK, from Bacillus subtilis is cold-activated through specific interhelical rearrangements in its central four-helix bundle domain. As revealed by the crystal structures of DesK in different functional states, the plasticity of this helical domain influences the catalytic activities of the protein, either by modifying the mobility of the ATP-binding domains for autokinase activity or by modulating binding of the cognate response regulator to sustain the phosphotransferase and phosphatase activities. The structural and biochemical data suggest a model in which the transmembrane sensor domain of DesK promotes these structural changes through conformational signals transmitted by the membrane-connecting two-helical coiled-coil, ultimately controlling the alternation between output autokinase and phosphatase activities. The structural comparison of the different DesK variants indicates that incoming signals can take the form of helix rotations and asymmetric helical bends similar to those reported for other sensing systems, suggesting that a similar switching mechanism could be operational in a wide range of sensor histidine kinases.

Molecular Recognition and Interfacial Catalysis by the Essential Phosphatidylinositol Mannosyltransferase PimA from Mycobacteria

Mycobacterial phosphatidylinositol mannosides (PIMs) and metabolically derived cell wall lipoglycans play important roles in host-pathogen interactions, but their biosynthetic pathways are poorly understood. Here we focus on Mycobacterium smegmatis PimA, an essential enzyme responsible for the initial mannosylation of phosphatidylinositol. The structure of PimA in complex with GDP-mannose shows the two-domain organization and the catalytic machinery typical of GT-B glycosyltransferases. PimA is an amphitrophic enzyme that binds mono-disperse phosphatidylinositol, but its transferase activity is stimulated by high concentrations of non-substrate anionic surfactants, indicating that the early stages of PIM biosynthesis involve lipid-water interfacial catalysis. Based on structural, calorimetric, and mutagenesis studies, we propose a model wherein PimA attaches to the membrane through its N-terminal domain, and this association leads to enzyme activation. Our results reveal a novel mode of phosphatidylinositol recognition and provide a template for the development of potential antimycobacterial compounds.

What Makes a Bacterial Species Pathogenic?:Comparative Genomic Analysis of the Genus Leptospira

Leptospirosis, caused by spirochetes of the genus Leptospira, is a globally widespread, neglected and emerging zoonotic disease. While whole genome analysis of individual pathogenic, intermediately pathogenic and saprophytic Leptospira species has been reported, comprehensive cross-species genomic comparison of all known species of infectious and non-infectious Leptospira, with the goal of identifying genes related to pathogenesis and mammalian host adaptation, remains a key gap in the field. Infectious Leptospira, comprised of pathogenic and intermediately pathogenic Leptospira, evolutionarily diverged from non-infectious, saprophytic Leptospira, as demonstrated by the following computational biology analyses: 1) the definitive taxonomy and evolutionary relatedness among all known Leptospira species; 2) genomically-predicted metabolic reconstructions that indicate novel adaptation of infectious Leptospira to mammals, including sialic acid biosynthesis, pathogen-specific porphyrin metabolism and the first-time demonstration of cobalamin (B12) autotrophy as a bacterial virulence factor; 3) CRISPR/Cas systems demonstrated only to be present in pathogenic Leptospira, suggesting a potential mechanism for this clade’s refractoriness to gene targeting; 4) finding Leptospira pathogen-specific specialized protein secretion systems; 5) novel virulence-related genes/gene families such as the Virulence Modifying (VM) (PF07598 paralogs) proteins and pathogen-specific adhesins; 6) discovery of novel, pathogen-specific protein modification and secretion mechanisms including unique lipoprotein signal peptide motifs, Sec-independent twin arginine protein secretion motifs, and the absence of certain canonical signal recognition particle proteins from all Leptospira; and 7) and demonstration of infectious Leptospira-specific signal-responsive gene expression, motility and chemotaxis systems. By identifying large scale changes in infectious (pathogenic and intermediately pathogenic) vs. non-infectious Leptospira, this work provides new insights into the evolution of a genus of bacterial pathogens. This work will be a comprehensive roadmap for understanding leptospirosis pathogenesis. More generally, it provides new insights into mechanisms by which bacterial pathogens adapt to mammalian hosts.

Structural and Kinetic Analysis of Two Covalent Sialosyl-Enzyme Intermediates on Trypanosoma rangeli Sialidase

Trypanosoma rangeli sialidase is a glycoside hydrolase (family GH33) that catalyzes the cleavage of α-2→3-linked sialic acid residues from sialoglycoconjugates with overall retention of anomeric configuration. Retaining glycosidases usually operate through a ping-pong mechanism, wherein a covalent intermediate is formed between the carbohydrate and an active site carboxylic acid of the enzyme. Sialidases, instead, appear to use a tyrosine as the catalytic nucleophile, leaving the possibility of an essentially different catalytic mechanism. Indeed, a direct nucleophilic role for a tyrosine was shown for the homologous trans-sialidase from Trypanosoma cruzi, although itself not a typical sialidase. Here we present the three-dimensional structures of the covalent glycosyl-enzyme complexes formed by the T. rangeli sialidase with two different mechanism-based inactivators at 1.9 and 1.7Å resolution. To our knowledge, these are the first reported structures of enzymatically competent covalent intermediates for a strictly hydrolytic sialidase. Kinetic analyses have been carried out on the formation and turnover of both intermediates, showing that structural modifications to these inactivators can be used to modify the lifetimes of covalent intermediates. These results provide further evidence that all sialidases likely operate through a similar mechanism involving the transient formation of a covalently sialylated enzyme. Furthermore, we believe that the ability to “tune” the inactivation and reactivation rates of mechanism-based inactivators toward specific enzymes represents an important step toward developing this class of inactivators into therapeutically useful compounds.

Structural insights into sialic acid enzymology

Sialic acids are a diverse family of negatively charged sugars that play essential biological roles. Their presence and relative abundance in different cells is ultimately regulated by the concerted action of a large set of enzymes. In this review, we focus on the most recent advances on the enzymes that govern sialic acid metabolism, with emphasis on structural work. Major progress has been made in dissecting the catalytic mechanism of sialidases, revealing a modified scenario of the typical glycosidase ping-pong mechanism. Similarly, X-ray structures of sialyltransferases uncover significant variations of formerly known glycosyltransferase foldings. Both sialidases and sialyltransferases seem to tell us that sialic acid-handling enzymes have evolved important modifications related to the distinctive features of sialic acid itself.