Structural Biology of Legionella pneumophila Effectors

Legionella pneumophila is a Gram-negative intracellular pathogen that causes Legionnaires’ disease and Pontiac fever in elderly or immunocompromised humans. The ability of Legionella to thrive within host cells depends on the Legionella-containing vacuole (LCV) which, in turn, relies on the activity of secreted effector proteins for its formation. Effectors are highly variable in structure and function, and functional redundancy is prevalent among them. Consequently, relating structural data to function provides an attractive avenue of research into molecules which are unlikely to exhibit a phenotype upon gene deletion. Our lab relies on X-ray crystallography for macromolecular structure determination. Structural data may point to a function for the protein of interest, which can be verified using mutagenesis, biochemical assays or some combination thereof. This dissertation explores the structure and putative function of effectors LpnE(lpg2222), MavE(lpg2344) and MavL(lpg2526). LpnE (Legionella pneumophila Entry) is a Sel1-like repeat (SLR) protein implicated in host cell invasion. During infection, a eukaryotic polyphosphate 5-phosphatase, called Oculocerebrorenal syndrome of Lowe protein 1 (OCRL1), is recruited to the LCV by an interaction with LpnE and restricts bacterial replication by an unknown mechanism. The crystal structure of His-LpnE(73-375) reveals a typical SLR super-helix with a concave surface implicated in protein-protein interactions. Herein, critical residues promoting the LpnE-OCRL interaction are uncovered using size exclusion chromatography with multi-angle light scattering (SEC-MALS). In addition, we show that LpnE localizes to cis¬-Golgi using its signal peptide. These findings are compiled into a mechanistic hypothesis where: (1) LpnE localizes to the LCV by its predicted signal peptide. (2) OCRL binding prevents liberation of LpnE from the LCV and (3) renders LpnE unable to promote infection by mediating protein-protein interactions in the cytosol. MavE is one of many proteins identified as a secreted effector based on its ability to rescue LCV localization of a translocation deficient SidC (SidCΔ100). Our collaborator, Dr. Yousef Abu-Kwaik, has obtained a unique phenotype for Δlpg2344 (MavE) mutants, in which the LCV fuses with lysosomes (unpublished data). He suggests that MavE interacts with proteins harbouring phosphotyrosine-binding domains (PTBs) using its NPxY motif. The recruitment of these binding partners may impede autophagic trafficking. The crystal structure of MavE(39-172) presented in this dissertation has an overall structure reminiscent of the grass pollen allergen, Phlp 5b, with the NPxY motif located on a loop of poorly defined electron density. This loop has no counterpart in Phlp 5b and has flexibility that may accommodate protein-protein interactions. These structural data corroborate the proposed role of the NPxY motif while revealing a scaffold domain previously seen in the grass pollen allergen, Phlp 5b. MavL is another secreted effector identified in the same manner as MavE. Presently, there is little published data available on the function of MavL. Elizabeth Hartland et al. have found by yeast two-hybrid that an E2 ubiquitin conjugating enzyme called Ube2q1 interacts directly with MavL, although we were unable to reproduce this interaction in vitro. The crystal structure of MavL(42-435) reveals an ADP-ribose binding macrodomain with homology to those that recognize mono-ADP-ribosylated targets. We confirmed the interaction of MavL and ADP-ribose by isothermal titration calorimetry (ITC), giving a dissociation constant of 13µM. Intriguingly, MavL contains a pair of neighboring aspartate residues in the same location as the catalytic glutamates of poly-ADP-ribose glycohydrolase (PARG) enzymes. We propose that MavL exhibits either ADP-ribose reader or eraser activity. Further studies are needed to investigate the role of ADP-ribosylation in MavL functionality

ACME algorithms for contact in a multiphysics environment API version 2.2.

An effort is underway at Sandia National Laboratories to develop a library of algorithms to search for potential interactions between surfaces represented by analytic and discretized topological entities. This effort is also developing algorithms to determine forces due to these interactions for transient dynamics applications. This document describes the Application Programming Interface (API) for the ACME (Algorithms for Contact in a Multiphysics Environment) library

Structural and functional characterization of Legionella pneumophila effector MavL

Abstract: Legionella pneumophila is a Gram-negative intracellular pathogen that causes Legionnaires' disease in elderly or immunocompromised individuals. This bacterium relies on the Dot/Icm (Defective in organelle trafficking/Intracellular multiplication) Type IV Secretion System (T4SS) and a large (>330) set of effector proteins to colonize the host cell. The structural variability of these effectors allows them to disrupt many host processes. Herein, we report the crystal structure of MavL to 2.65 Å resolution. MavL adopts an ADP-ribosyltransferase (ART) fold and contains the distinctive ligand-binding cleft of ART proteins. Indeed, MavL binds ADP-ribose with Kd of 13 µM. Structural overlay of MavL with poly-(ADP-ribose) glycohydrolases (PARGs) revealed a pair of aspartate residues in MavL that align with the catalytic glutamates in PARGs. MavL also aligns with ADP-ribose “reader” proteins (proteins that recognize ADP-ribose). Since no glycohydrolase activity was observed when incubated in the presence of ADP-ribosylated PARP1, MavL may play a role as a signaling protein that binds ADP-ribose. An interaction between MavL and the mammalian ubiquitinconjugating enzyme UBE2Q1 was revealed by yeast two-hybrid and co-immunoprecipitation experiments. This work provides structural and molecular insights to guide biochemical studies aimed at elucidating the function of MavL. Our findings support the notion that ubiquitination and ADP-ribosylation are global modifications exploited by L. pneumophila

Computational Studies in Molecular Geochemistry and Biogeochemistry

The ability to predict the transport and transformations of contaminants within the subsurface is critical for decisions on virtually every waste disposal option facing the Department of Energy (DOE), from remediation technologies such as in situ bioremediation to evaluations of the safety of nuclear waste repositories. With this fact in mind, the DOE has recently sponsored a series of workshops on the development of a Strategic Simulation Plan on applications of high perform-ance computing to national problems of significance to the DOE. One of the areas selected for application was in the area of subsurface transport and environmental chemistry. Within the SSP on subsurface transport and environmental chemistry several areas were identified where applications of high performance computing could potentially significantly advance our knowledge of contaminant fate and transport. Within each of these areas molecular level simulations were specifically identified as a key capability necessary for the development of a fundamental mechanistic understanding of complex biogeochemical processes. This effort consists of a series of specific molecular level simulations and program development in four key areas of geochemistry/biogeochemistry (i.e., aqueous hydrolysis, redox chemistry, mineral surface interactions, and microbial surface properties). By addressing these four differ-ent, but computationally related, areas it becomes possible to assemble a team of investigators with the necessary expertise in high performance computing, molecular simulation, and geochemistry/biogeochemistry to make significant progress in each area. The specific targeted geochemical/biogeochemical issues include: Microbial surface mediated processes: the effects of lipopolysacchardies present on gram-negative bacteria. Environmental redox chemistry: Dechlorination pathways of carbon tetrachloride and other polychlorinated compounds in the subsurface. Mineral surface interactions: Describing surfaces at multiple scales with realistic surface functional groups Aqueous Hydrolysis Reactions and Solvation of Highly Charged Species: Understanding the formation of polymerized species and ore formation under extreme (Hanford Vadose Zone and geothermo) conditions. By understanding on a fundamental basis these key issues, it is anticipated that the impacts of this research will be extendable to a wide range of biogeochemical issues. Taken in total such an effort truly represents a “Grand Challenge” in molecular geochemistry and biogeochemistry

An Indispensable Role for the MavE Effector of in Lysosomal Evasion

ABSTRACT Diversion of the -containing vacuole (LCV) from the host endosomal-lysosomal degradation pathway is one of the main virulence features essential for manifestation of Legionnaires’ pneumonia. Many of the ∼350 Dot/Icm-injected effectors identified in have been shown to interfere with various host pathways and processes, but no effector has ever been identified to be indispensable for lysosomal evasion. While most single effector mutants of do not exhibit a defective phenotype within macrophages, we show that the MavE effector is essential for intracellular growth of in human monocyte-derived macrophages (hMDMs) and amoebae and for intrapulmonary proliferation in mice. The null mutant fails to remodel the LCV with endoplasmic reticulum (ER)-derived vesicles and is trafficked to the lysosomes where it is degraded, similar to formalin-killed bacteria. During infection of hMDMs, the MavE effector localizes to the poles of the LCV membrane. The crystal structure of MavE, resolved to 1.8 Å, reveals a C-terminal transmembrane helix, three copies of tyrosine-based sorting motifs, and an NPxY eukaryotic motif, which binds phosphotyrosine-binding domains present on signaling and adaptor eukaryotic proteins. Two point mutations within the NPxY motif result in attenuation of in both hMDMs and amoeba. The substitution defects of P and D are associated with failure of vacuoles harboring the mutant to be remodeled by the ER and results in fusion of the vacuole to the lysosomes leading to bacterial degradation. Therefore, the MavE effector of is indispensable for phagosome biogenesis and lysosomal evasion. Intracellular proliferation of within a vacuole in human alveolar macrophages is essential for manifestation of Legionnaires’ pneumonia. Intravacuolar growth of the pathogen is totally dependent on remodeling the -containing vacuole (LCV) by the ER and on its evasion of the endosomal-lysosomal degradation pathway. The pathogen has evolved to inject ∼350 protein effectors into the host cell where they modulate various host processes, but no effector has ever been identified to be indispensable for lysosomal evasion. We show that the MavE effector localizes to the poles of the LCV membrane and is essential for lysosomal evasion and intracellular growth of and for intrapulmonary proliferation in mice. The crystal structure of MavE shows an NPxY eukaryotic motif essential for ER-mediated remodeling and lysosomal evasion by the LCV. Therefore, the MavE effector of is indispensable for phagosome biogenesis and lysosomal evasion