13 research outputs found
Hijacking natureânew approaches to unravel enzyme mechanisms and engineer improved biocatalysts
Open and closed conformations of a sub-80 kDa Chagas vaccine candidate defined by a cryo-EM led integrative approach
Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, remains a significant global public health concern. It affects an estimated eight million individuals worldwide, with the majority remaining undiagnosed. Despite its profound health impact in both endemic and non-endemic areas, no vaccine is available, and the existing therapies are outdated, producing severe side effects.The 80kDa prolyl oligopeptidase of Trypanosoma cruzi (TcPOP) has been recently identified as a leading candidate for Chagas vaccine development. However, its three-dimensional structure has remained elusive for almost two decades since its discovery. We report the first three-dimensional structure of TcPOP in open and closed conformation, at a resolution of 3.0 and 2.5 Angstroms respectively, determined using single-particle cryo-electron microscopy. Multiple conformations were observed and were further characterized, using plasmonic optical tweezers.To assess the immunogenic potential of TcPOP, we immunized mice and evaluated both polyclonal and monoclonal responses against the TcPOP antigen and its homologues. The results revealed unexpected cross-reactivity across prolyl POPs from other closely related parasites, but intriguingly, not towards the human homologue.Altogether, our findings provide critical structural insights necessary to understand the immunogenicity of TcPOP for future Chagas vaccine development and diagnostic applications.Additionally, our integrative approach indicated that stage-tilted acquisition can yield biologically relevant information for challenging sub-80kDa proteins and could adequately resolve the cryoEM structures. Consequently, this comprehensive strategy can significantly enhance the success rate in determining the structures of proteins that present challenges in characterization
The Chlamydia effector TarP mimics the mammalian leucine-aspartic acid motif of paxillin to subvert the focal adhesion kinase during invasion
Host cell signal transduction pathways are often targets of bacterial pathogens, especially during the process of invasion when robust actin remodeling is required. We demonstrate that the host cell focal adhesion kinase (FAK) was necessary for the invasion by the obligate intracellular pathogen Chlamydia caviae. Bacterial adhesion triggered the transient recruitment of FAK to the plasma membrane to mediate a Cdc42- and Arp2/3-dependent actin assembly. FAK recruitment was via binding to a domain within the virulence factor TarP that mimicked the LD2 motif of the FAK binding partner paxillin. Importantly, bacterial two-hybrid and quantitative imaging assays revealed a similar level of interaction between paxillin-LD2 and TarP-LD. The conserved leucine residues within the L(D/E)XLLXXL motif were essential to the recruitment of FAK, Cdc42, p34Arc, and actin to the plasma membrane. In the absence of FAK, TarP-LD-mediated F-actin assembly was reduced, highlighting the functional relevance of this interaction. Together, the data indicate that a prokaryotic version of the paxillin LD2 domain targets the FAK signaling pathway, with TarP representing the first example of an LD-containing Type III virulence effector
Structural analysis of engineered N-acetylneuraminic acid lyase mutants
EThOS - Electronic Theses Online ServiceGBUnited Kingdo
The Potential of Plant-Produced Virus-like Particle Vaccines for African Horse Sickness and Other Equine Orbiviruses
African horse sickness is a devastating viral disease of equids. It is transmitted by biting midges of the genus Culicoides with mortalities reaching over 90% in naĂŻve horses. It is endemic to sub-Saharan Africa and is seasonally endemic in many parts of southern Africa. However, outbreaks in Europe and Asia have occurred that caused significant economic issues. There are attenuated vaccines available for control of the virus but concerns regarding the safety and efficacy means that alternatives are sought. One promising alternative is the use of virus-like particles in vaccine preparations, which have the potential to be safer and more efficacious as vaccines against African horse sickness. These particles are best made in a complex, eukaryotic system, but due to technical challenges, this may cause significant economic strain on the developing countries most affected by the disease. Therefore, this review also summarises the success so far, and potential, of recombinant protein expression in plants to reduce the economic strain of production
Structure of an Escherichia coli N-acetyl-d-neuraminic acid lyase mutant, E192N, in complex with pyruvate at 1.45â Ă resolution
The 1.45â
Ă
structure of E. coli N-acetyl-d-neuraminic acid lyase in complex with pyruvate in space group P212121 is reported from new low-salt crystallization conditions that will facilitate soaking experiments with substrates and inhibitors
Structural Insights into Substrate Specificity in Variants of N-Acetylneuraminic Acid Lyase Produced by Directed Evolution
The substrate specificity of Escherichia coli N-acetylneuraminic acid lyase was previously switched from the natural condensation of pyruvate with N-acetylmannosamine, yielding N-acetylneuraminic acid, to the aldol condensation generating N-alkylcarboxamide analogues of N-acetylneuraminic acid. This was achieved by a single mutation of Glu192 to Asn. In order to analyze the structural changes involved and to more fully understand the basis of this switch in specificity, we have isolated all 20 variants of the enzyme at position 192 and determined the activities with a range of substrates. We have also determined five high-resolution crystal structures: the structures of wild-type E. coli N-acetylneuraminic acid lyase in the presence and in the absence of pyruvate, the structures of the E192N variant in the presence and in the absence of pyruvate, and the structure of the E192N variant in the presence of pyruvate and a competitive inhibitor (2R,3R)-2,3,4-trihydroxy-N,N-dipropylbutanamide. All structures were solved in space group P21 at resolutions ranging from 1.65Â Ă
to 2.2Â Ă
. A comparison of these structures, in combination with the specificity profiles of the variants, reveals subtle differences that explain the details of the specificity changes. This work demonstrates the subtleties of enzymeâsubstrate interactions and the importance of determining the structures of enzymes produced by directed evolution, where the specificity determinants may change from one substrate to another
One-step design of a stable variant of the malaria invasion protein RH5 for use as a vaccine immunogen
Many promising vaccine candidates from pathogenic viruses, bacteria, and parasites are unstable and cannot be produced cheaply for clinical use. For instance, Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is essential for erythrocyte invasion, is highly conserved among field isolates, and elicits antibodies that neutralize in vitro and protect in an animal model, making it a leading malaria vaccine candidate. However, functional RH5 is only expressible in eukaryotic systems and exhibits moderate temperature tolerance, limiting its usefulness in hot and low-income countries where malaria prevails. Current approaches to immunogen stabilization involve iterative application of rational or semirational design, random mutagenesis, and biochemical characterization. Typically, each round of optimization yields minor improvement in stability, and multiple rounds are required. In contrast, we developed a one-step design strategy using phylogenetic analysis and Rosetta atomistic calculations to design PfRH5 variants with improved packing and surface polarity. To demonstrate the robustness of this approach, we tested three PfRH5 designs, all of which showed improved stability relative to wild type. The best, bearing 18 mutations relative to PfRH5, expressed in a folded form in bacteria at >1 mg of protein per L of culture, and had 10-15 °C higher thermal tolerance than wild type, while also retaining ligand binding and immunogenic properties indistinguishable from wild type, proving its value as an immunogen for a future generation of vaccines against the malaria blood stage. We envision that this efficient computational stability design methodology will also be used to enhance the biophysical properties of other recalcitrant vaccine candidates from emerging pathogens
Protein-to-structure pipeline for ambient-temperature in situ crystallography at VMXi
The utility of X-ray crystal structures determined under ambient-temperature conditions is becoming increasingly recognized. Such experiments can allow protein dynamics to be characterized and are particularly well suited to challenging protein targets that may form fragile crystals that are difficult to cryo-cool. Room-temperature data collection also enables time-resolved experiments. In contrast to the high-throughput highly automated pipelines for determination of structures at cryogenic temperatures widely available at synchrotron beamlines, room-temperature methodology is less mature. Here, the current status of the fully automated ambient-temperature beamline VMXi at Diamond Light Source is described, and a highly efficient pipeline from protein sample to final multi-crystal data analysis and structure determination is shown. The capability of the pipeline is illustrated using a range of user case studies representing different challenges, and from high and lower symmetry space groups and varied crystal sizes. It is also demonstrated that very rapid structure determination from crystals in situ within crystallization plates is now routine with minimal user intervention
Reaction Mechanism of <i>N</i>âAcetylneuraminic Acid Lyase Revealed by a Combination of Crystallography, QM/MM Simulation, and Mutagenesis
<i>N</i>-Acetylneuraminic acid lyase (NAL) is a Class I aldolase
that catalyzes the reversible condensation of pyruvate with <i>N</i>-acetyl-d-mannosamine (ManNAc) to yield the sialic
acid <i>N</i>-acetylneuraminic acid (Neu5Ac). Aldolases
are finding increasing use as biocatalysts for the stereospecific
synthesis of complex molecules. Incomplete understanding of the mechanism
of catalysis in aldolases, however, can hamper development of new
enzyme activities and specificities, including control over newly
generated stereocenters. In the case of NAL, it is clear that the
enzyme catalyzes a Bi-Uni ordered condensation reaction in which pyruvate
binds first to the enzyme to form a catalytically important Schiff
base. The identity of the residues required for catalysis of the condensation
step and the nature of the transition state for this reaction, however,
have been a matter of conjecture. In order to address, this we crystallized
a Y137A variant of the <i>E. coli</i> NAL in the presence
of Neu5Ac. The three-dimensional structure shows a full length sialic
acid bound in the active site of subunits A, B, and D, while in subunit
C, discontinuous electron density reveals the positions of enzyme-bound
pyruvate and ManNAc. These âsnapshotâ structures, representative
of intermediates in the enzyme catalytic cycle, provided an ideal
starting point for QM/MM modeling of the enzymic reaction of carbonâcarbon
bond formation. This revealed that Tyr137 acts as the proton donor
to the aldehyde oxygen of ManNAc during the reaction, the activation
barrier is dominated by carbonâcarbon bond formation, and proton
transfer from Tyr137 is required to obtain a stable Neu5Ac-Lys165
Schiff base complex. The results also suggested that a triad of residues,
Tyr137, Ser47, and Tyr110 from a neighboring subunit, are required
to correctly position Tyr137 for its function, and this was confirmed
by site-directed mutagenesis. This understanding of the mechanism
and geometry of the transition states along the CâC bond-forming
pathway will allow further development of these enzymes for stereospecific
synthesis of new enzyme products