Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, can evade host immune response. Peroxidases, ferritins, and folate aldolases are antioxidant enzymes, protecting Mtb from oxidative damage by reactive oxygen species in activated macrophages. Dye-decolorizing peroxidases (DyPs) are encapsulated by a bacterial nanocompartment, encapsulin (Enc), where packaged DyP interacts with Enc via a unique C-terminal extension. Genes encoding for Enc exist in an operon with DyPs. Mtb harbors an encapsulin homolog (Mt-Enc) adjacent to the gene that encodes for Mt-DyP. Our studies indicate that Mt-Enc self-assembles into a 60-subunit icosahedral shell independently of cargo enzymes. Preliminary characterization suggests that Mt-DyP is a heme-peroxidase with a C-terminal extension important for interaction with Mt-Enc--elimination of the C-terminal extension abolishes the interaction.

Mtb iron-storage ferritin protein (Mt-BfrB) and 7,8-dihydroneopterin aldolase (Mt-FolB) have C-terminal tails that also interact with Mt-Enc. Mt-DyP and Mt-BfrB are predicted to be involved in oxidative stress response. Mt-FolB may mitigate oxidative attack, where Mt-FolB allows 7,8-dihydroneopterin to accumulate acting as an antioxidant. We observed encapsulation of Mt-DyP, Mt-BfrB, and Mt-FolB via co-purification and electron microscopy. Protein encapsulation has not been observed in vivo and its purpose is currently unknown, specifically encapsulation of enzymes involved in oxidative stress response. Functional studies of these three enzymes while encapsulated demonstrate that these enzymes retain enzymatic activity: Mt-DyP exhibits peroxidase activity with ABTS as a substrate, Mt-BfrB exhibits ferroxidase activity, and Mt-FolB exhibits aldolase activity; however, Mt-Enc alone does not appear to have enzymatic activity. This is the first study that illustrates mycobacterial encapsulation, providing structural and biochemical clues about mechanisms by which Mtb can detoxify the local environment to ensure long-term survival. We have also begun structural studies, probing Mt-Enc nanocompartment assembly in order to understand formation and utilize Mt-Enc as a therapeutic delivery platform.

In vivo analyses elucidating the role of Mt-Enc, and its involvement in survival during oxidative stress have started. Here, we aim to: i) characterize Mt-Enc; ii) preliminarily characterize DyP as a peroxidase; iii) begin structural analyses of both Mt-Enc and Mt-DyP; and iv) begin studies to determine the role of Mt-Enc and its implications in mycobacterial survival.

Iron is important for pathogen survival and pathogens such as Mycobacterium tuberculosis (Mtb) have evolved pathways to acquire host iron. One such mechanism Mtb has evolved is the acquisition of heme, where Mtb imports host heme for degradation and iron release. In the cytosol, the heme degrading enzyme MhuD is responsible for the cleavage of heme and the liberation iron for use by Mtb. In this work we document our ongoing efforts to better understand MhuD and its larger role in Mtb.

Although the structure of the inactive diheme form of MhuD was previously determined, the structure of its active monoheme form was unresolved. We were able to determine the crystal structure of the cyano-derivative of MhuD-monoheme to obtain insights into MhuD heme degradation and product uniqueness. Heme ruffling, proposed to be responsible for heme’s reactivity within the active site of MhuD homologs, was observed in the MhuD-monoheme structure. Mutations to apparent heme ruffling residues Phe23 and Trp66 appear to abolish MhuD’s heme degradation activity suggesting the importance of heme ruffling in MhuD activity. Residues were also identified potentially responsible for MhuD’s unique degradation products. We found that mutation of one such residue, Arg26 into a serine, alters MhuD’s heme degradation into biliverdin and formaldehyde. Efforts to biophysically and structurally characterize the MhuD R26S mutant suggests that there is a reduction in heme ruffling in the mutant which may lead to the altered degradation product.

We also explored the role MhuD plays in the pharmacological inhibition of Mtb growth of mice infected with Mtb by the heme analog tin-protoporphrin IX (SnPPIX). Despite its ability to inhibit the human heme degrader, heme oxygenase, SnPPIX did not appear to inhibit MhuD’s ability to degrade heme. Lastly, we also carried out preliminary research towards the identification of a MhuD electron donor as well as of a potential protein partner for MhuD product release. To date, neither proposed MhuD interaction partners have been identified, however more candidate proteins remain to be purified and tested.

Bacteria have developed complex mechanisms to thrive in their environments. Contact- dependent growth inhibition (CDI), a novel inter-bacterial competition mechanism was recently discovered in gram-negative bacteria, and is used to block growth of neighboring cells. The CDI growth inhibitory activity is contained in the C-terminal domain of CdiA effector proteins (CdiA- CT, toxin), and is delivered into target bacterial cells. All CDI+ bacteria also produce small CdiI immunity proteins that protect them from autoinhibition. CdiA-CT and CdiI from different species exhibit high sequence variability, which predicts diverse structures and/or interaction interfaces. Here we describe the initial structural characterization of the CDI system, providing functional insights into distinct CDI mechanisms.

We have solved X-ray crystal structures of CDI toxin/immunity complexes from Escherichia coli (EC869), Burkholderia pseudomallei (1026b), and Enterobacter cloacae (ECL), representing three distinct families of CDI proteins. The interfaces between the toxin/immunity pairs are distinct, providing important insights into CdiA-CT/CdiI binding specificity. The EC869 CdiA-CT/CdiI binding interface is especially intriguing and consists of a unique β-augmentation interaction, by which CdiA-CT^EC869 inserts a short β-hairpin structural element into a cavity on the surface of CdiI^EC869. This interface is conserved among EC869 family CdiA-CT/CdiI complexes, but heterologous toxin and immunity proteins within this family do not interact with high affinity. Additionally, the EC869, 1026b, and ECL toxin structures helped elucidate and characterize three distinct CdiA-CT growth-inhibiting activities. EC869 and 1026b CdiA-CTs are structurally similar and contain DNase and tRNase activities, respectively. In contrast, ECL CdiA-CT shares no structural homology with EC869 or 1026b CdiA-CTs, but is structurally and functionally similar to colicin E3, a toxin with rRNase activity from a distinct bacterial system. We also report the crystal structure of the CdiA-CT toxin from uropathogenic E. coli strain 536 in complex with its permissive factor, CysK, representing a unique step in CDI. The complex formed mimics the interaction found in the cysteine synthase complex. Finally, we solved the structure of a domain from the large, non-toxin region of E. coli CdiA, providing a starting point towards future research into the CdiA-CT cleavage and translocation mechanisms.

Bacteria have developed complex mechanisms to survive and propagate within their environments. Contact-dependent growth inhibition (CDI) is a recently discovered mechanism of inter-bacterial competition and communication widespread amongst Gram-negative bacteria. CDI+ cells inhibit the growth of neighboring cells upon delivery of a toxic protein (CdiA-CT) into the target cell cytosol. To prevent auto-inhibition, CDI+ bacteria express an immunity protein (CdiI) to specifically bind and inactivate cognate toxin. Here we provide further structural characterization of CDI toxin/immunity proteins to elucidate the diversity of CdiA-CT toxin activity, their potential activation, and neutralization to repurpose CDI systems for novel antimicrobials.

Burkholderia pseudomallei isolates E479 and 1026b have been shown to utilize CDI to outcompete neighboring bacteria, signifying the pathway’s importance in growth and survival. The CdiA-CT toxins from these isolates are functional tRNases, yet have unique tRNA specificities and cleavage sites despite sharing no sequence similarity. We have solved the X-ray crystal structure of the B. pseudomallei E479 toxin/immunity protein complex and compared it to the previously solved 1026b complex. While the toxins are sequentially and functionally different, they share significant structural homology with nearly identical active sites, only differing in active site pocket size, correlating with the differing substrate cleavage sites of the two toxins. The toxin/tRNA interactions have been confirmed via molecular docking as well as small angle X-ray scattering.

The CDI system from uropathogenic E. coli 536 (UPEC536) is unusual, CdiA-CT exhibits toxic tRNase activity only when bound to target cell CysK, termed a “permissive factor”, involved in cysteine biosynthesis. We have solved the X-ray crystal structure of the CysK/toxin/immunity ternary complex. The UPEC536 toxin inserts its C-terminus into the active site cleft of CysK, mimicking its natural interaction with CysE. A homologous toxin from the Gram-positive Ruminococcus lactaris has been identified to perform the same enzymatic function in a CysK-independent manner. Characterization of this toxin has shown that it’s significantly more thermostable than the UPEC toxin, and is capable of binding tRNA substrate in the absence of CysK. This demonstrates a chaperone-like function for CysK, stabilizing the UPEC toxin fold and promoting its association with tRNA substrate.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, poses a great threat to human health. With the emergence of drug resistant Mtb strains, new therapeutics are desperately needed. As iron is critical to the growth and survival of Mtb, the mechanisms through which Mtb acquires host iron pose attractive therapeutic targets. Mtb scavenges iron from the host via Mtb siderophores, carboxymycobactin and mycobactin, and heme uptake. Heme uptake culminates in degradation by the cytosolic protein MhuD, a noncanonical heme degrading enzyme in the IsdG family, in Mtb. In a recent study, a product-bound structure of the MhuD-R26S mutant with ⍺-biliverdin was determined and revealed the formation of a novel secondary structural element, ⍺3, stabilized by the 75HisXXXR79 motif. In Chapter 2 I investigate the biological relevance of this motif by examining the heme binding and degradation of a MhuD R79S variant. I also determined that IsdI, another IsdG-family protein, binds with high affinity to the β- and δ-biliverdin isomers which will enable structural studies of product-bound IsdI. The fate of the heme breakdown product generated by MhuD, mycobilin, remained unknown. In Chapter 3, I demonstrate that Rv2074 is a novel mycobilin reductase and determined the chemical structure of the novel reduced mycobilin product, coined “mycorubin,” utilizing tandem mass spectrometry. Little is known regarding the transport of siderophores across the periplasm and cell-wall environment, but the Mtb periplasmic binding proteins FecB and FecB2 have been implicated in host iron acquisition. In Chapter 4 in vitro ligand binding experiments and structural comparisons for FecB and FecB2 were performed, revealing that both FecB and FecB2 bind heme, while only FecB binds Fe-cMB. Subsequent structure-guided mutagenesis of the FecB ligand binding site identified a single glutamate residue—Glu339—that significantly contributes to Fe-cMB binding. In Chapter 5, a role for FecB in the Mtb siderophore-mediated iron acquisition pathway was corroborated by Mycobacterium smegmatis pull-down experiments, which revealed interactions between FecB and known members of the mycobacterial siderophore export and import machinery, like MmpS5, a protein involved in apo-siderophore efflux. Finally, the FecB interacting partner MmpS5 was confirmed in Mtb by co-immunoprecipitation.

For decades, tuberculosis (TB) has persisted as a global health burden with over a million people succumbing to the disease each year. As cases of multi-drug resistant and extensively drug-resistant increase, there is an urgent need for the development of novel therapeutics to combat the disease. Fortunately, advances in protein structure determination and development of computational modeling tools provide valuable insight into the specific interactions the mediate binding between proteins and ligands. Nonetheless, the results of these methods are sometimes ambiguous or inconclusive. Taken together, structural determination and computational modeling can complement each other to provide more robust and interpretable data. In Chapter 2 of this work, I focus on the development of a computational method to enhance sampling of side chains, which can rearrange in the presence of different ligands. Specifically, I describe the development and validation of a non-equilibrium candidate Monte Carlo method for the enhanced sampling of side chain rotamers during molecular dynamics (MD) simulations. I validate this method on a simple valine-alanine dipeptide and demonstrate that it significantly improves rotamer sampling of a buried valine sidechain in the binding site of model system T4 lysozyme L99A. In Chapters 3 and 4, we use X-ray crystallography and MD to elucidate and study the structures of two potential TB drug targets from Mycobacterium tuberculosis (Mtb): Mtb heme oxygenase (MhuD) and Mtb malic enzyme (MEZ). In the structure of MhuD in complex with product, we observe the formation of a novel α-helix; however, the unusual 5:2 ratio of product to protein subunit in the asymmetric unit, as well as the proximity of the helix to the crystallographic interface, provoke questions regarding its biological relevance. Using MD, I confirm that formation of the α-helix is favored in the presence of product and likely associated with product-turnover. In Chapter 3, I describe the X-ray structure of apo-MEZ along with results from differential scanning fluorimetry and gel filtration. MD simulations provide insight into interactions of MEZ with NAD(P)+, Mn2+, and malate, while also corroborating our empirical observations that MEZ is unusually disordered.