<p>The posttranslation modification of histone proteins within the nucleosomes of chromatin plays important roles in the regulation of gene expression in both normal biological and pathobiological processes. These modifications alter local chromatin structure and subsequently alter the expression profile of associated genes. Histone methylation, which was long thought immutable, is one such modification that plays a dual functionality in both activation and repression of gene expression and can be thought of as an information storage mark. With the initial discovery of lysine-specific demethylase 1A (LSD1/KDM1A), an FAD-dependent enzyme that catalyzes the oxidative demethylation of histone H3K4me1/2 and H3K9me1/2 repressing and activating transcription, respectively, the missing counterbalance to dynamic histone methylation was cemented. This discovery further strengthened the link between histone demethylation and transcriptional regulation and the enzyme has since been identified as a target with therapeutic potential.</p><p>Given the significance of KDM1A enzymatic activity, herein we report our efforts to characterize novel binding interactions that dictate the enzymes biological and pathobiological functions. As KDM1A falls into the greater class of flavin-dependent amine oxidases, it contains features that are recurrent within the class, but due to its unique ability to work on histone and non-histone substrates has unprecedented structural elements. Although the active site is expanded compared to the greater amine oxidase superfamily, it is too sterically restricted to encompass the minimal 21-mer peptide substrate footprint of the histone H3 tail. The remainder of the substrate/product is therefore expected to extend along the surface of KDM1A. Using steady-state kinetic analyses, we now show that unmodified histone H3 is a tight-binding, competitive inhibitor of KDM1A demethylation activity with a Ki of 18.9 ± 1.2 nM that is approximately 100-fold higher than the 21-mer peptide product. The relative affinity of dose-response curves is independent of preincubation time suggesting that H3 rapidly reaches equilibrium with KDM1A. Rapid dilution experiments confirmed the increased binding affinity of full-length H3 toward KDM1A was at least partially caused by a slow off-rate with a koff of 0.072 min-1, a half-life (t1/2) of 9.63 min, and residence time (τ) of 13.9 min. Independent affinity capture surface plasmon resonance experiments confirmed the tight-binding nature of the H3/KDM1A interaction revealing a Kd of 9.02 ± 2.27 nM, a kon of 9.26 x 104 ± 1.5 x 104 M-1s-1 and koff of 8.35 x 10-4 ± 3.4 x 105 s-1. Additionally, consistent with H3 being the only histone substrate of KDM1A, no other core histones are inhibitors of demethylation activity. Our data suggests that KDM1A contains a histone H3 secondary specificity recognition element on the enzyme surface and required further characterization.</p><p>In order to characterize this secondary H3 binding site, we turned to the use of cysteine labeling, chemical cross-linking coupled to proteolysis and LC-MS/MS, HDX-MS, and the design of an active, tower domain deletion KDM1A mutant. We now show that the tower domain contributes to the extended binding interface of the KDM1A/H3 interaction. Additionally, we show that the KDM1A tower domain is not required for demethylation activity and that one can functionally uncouple catalytic activity from protein-protein interactions that occur along the KDM1A tower domain interface, a domain unprecedented in the greater amine oxidase family. Furthermore, this towerless mutant will be useful for dissecting molecular contributions to KDM1A function along the tower domain. Our discovery of this secondary binding site within the aforementioned domain points to how pivotal this region is to the control and localization of KDM1A enzymatic activity as it also serves a pivotal role as a protein-protein interaction motif for the nucleation of a multitude of multimeric protein complexes.</p><p>With this in mind, we set out to design a strategy to isolate the core histone demethylase complex from E. coli cellular lysates. With the use of polycistronic vectors that encode both KDM1A and CoREST for coexpression we were able to produce appreciable amounts of chromatographically pure complex. As our CoREST construct in this strategy contains both the ELM2 and SANT2 domain needed for interaction with the HDACs, this core complex will serve as a starting point for future work that will tease apart additional influences on substrate binding and recognition imparted on KDM1A from binding partners. This preparation can therefore be used in a multitude of downstream studies including reconstitution of the core histone demethylase/deactylase complex and in depth kinetic and biophysical analyses and provides an invaluable starting point</p><p>This work provides a foundational understanding of this unprecedented secondary binding site on the surface of the KDM1A tower domain and how it may play an important role in substrate and product recognition. We suspect that this extended interaction interface may control KDM1A localization within specific chromatin loci and allow the enzyme to serve as a docking element for the nucleation of protein complexes or transcriptional machinery. On the other hand, disruption of this point of contact between the KDM1A/H3 binary complex may also facilitate enzyme/product dissociation, thereby tuning the catalytic activity of the demethylase. Additionally, the ability to produce substantial quantities of the core histone demethylase complex is a necessary step in the decoding of the ‘histone code’ hypothesis of KDM1A and its associated complexes. We suspect that the body of this work will prove to be invaluable for future characterization of the enzyme and its role in biology and pathobiology.</p> / Dissertation

Bacterial DNA replication can stall at DNA lesions, leading to cell death if the damage fails to be repaired. To circumvent this, bacteria possess a mechanism called translesion DNA synthesis (TLS) to allow DNA damage bypass. The ImuABC TLS mutasome comprises the RecA domain-containing protein ImuA, the inactive polymerase ImuB, and the error-prone polymerase ImuC. ImuA and ImuB are necessary for the mutational function of ImuC that can lead to antimicrobial resistance (AMR) as seen in high-priority pathogens Pseudomonas aeruginosa and Mycobacterium tuberculosis. Understanding how ImuA and ImuB contribute to this function can lead to new targets for antimicrobial development. This research aims to discover the molecular functions of ImuA and ImuB homologs from Myxococcus xanthus through structural modelling and biochemical analyses. ImuA was discovered to be an ATPase whose activity is enhanced by DNA. Based on predicted structural models of the ATPase active site, I identified the critical residues needed for ATP hydrolysis, and found that the ImuA C-terminus regulates ATPase activity. Further, ImuA and ImuBNΔ34 (a soluble truncation of ImuB) display a preference for longer single-stranded DNA and overhang DNA substrates, and their affinity for DNA was quantified in vitro. To better understand how ImuA and ImuB assemble in the TLS mutasome, bacterial two-hybrid assays determined that ImuA and ImuB can self-interact and bind one another. Mass photometry revealed that ImuA is a monomer and ImuBNΔ34 is a trimer in vitro. ImuA and ImuBNΔ34 binding affinity was quantified in vitro at 1.69 μM ± 0.21 by microscale thermophoresis, and removal of the ImuA C-terminus weakens this interaction. Lastly, ImuA and ImuBNΔ34 secondary structures were quantified using circular dichroism spectroscopy, and ImuA was modified to enable crystallization for future structural studies. Together, this research provides a better understanding of ImuABC-mediated TLS, potentially leading to novel antibiotics to reduce the clinical burden of AMR. / Thesis / Master of Science (MSc) / The antimicrobial resistance (AMR) crisis is fueled by the emergence of multi-drug resistant microbes, posing a major threat to global health and disease treatment. Bacteria can develop resistance to antibiotics through mutations in the genome. When the genome becomes damaged, bacteria can acquire these mutations by an error-prone replication mechanism called translesion DNA synthesis (TLS). In some bacteria, TLS involves a specialized enzyme complex, consisting of proteins ImuA, ImuB and ImuC, allowing replication past bulky DNA damage and lesions. The goal of this thesis is to investigate how the ImuA and ImuB proteins contribute to the functioning of this mistake-making machinery. I used biochemical and biophysical methods to identify ImuA and ImuB interactions with each other and themselves. I discovered that ImuA is an enzyme that uses energy to enhance its binding to DNA, and determined the specific amino acids involved in this function.

Downstream regulatory element antagonist modulator (DREAM) is involved in various interactions with targets both inside and outside of the nucleus. In the cytoplasm, DREAM interacts with the C-terminal fragments of presenilins to facilitate the production of β-amyloid plaques in Alzheimer’s disease. In the nucleus, Ca2+ free DREAM directly binds to specific downstream regulatory elements of prodynorphin/c-fos gene to repress the gene transcription in pain modulation. These interactions are regulated by Ca2+ and/or Mg2+ association at the EF-hands in DREAM. Therefore, understanding the conformational dynamics and stability associated with Ca2+ and/or Mg2+ binding to DREAM is crucial for elucidating the mechanisms of interactions of DREAM with DNA or presenilins. The critical barrier for envisioning the mechanisms of these interactions lies in the lack of NMR/crystal structures of Apo and Mg2+DREAM. Using a combination of fluorescence spectroscopy, circular dichroism, isothermal titration calorimetry, photothermal spectroscopy, and computational approaches, I showed that Mg2+ association at the EF-hand 2 structurally stabilizes the N-terminal alpha-helices 1, 2, and 5, facilitating the interaction with DNA. Binding of Ca2+ at the EF-hand 3 induces significant structural changes in DREAM, mediated by several hydrophobic residues in both the N- and C-domains. These findings illustrate the critical role of EF-hand 3 for Ca2+ signal transduction from the C- to N-terminus in DREAM. The Ca2+ association at the EF-hand 4 stabilizes the C-terminus by forming a cluster consisting of several hydrophobic residues in C-terminal domain. I also demonstrated that association of presenilin-1 carboxyl peptide with DREAM is Ca2+ dependent and the complex is stabilized by aromatic residues F462 and F465 from presenilin-1 and F252 from DREAM. Stabilization is also provided by residues R200 and R207 in the loop connecting a7 and a8 in DREAM and the residues D450 and D458 in presenilin-1. These findings provide a structural basis for the development of new drugs for chronic pain and Alzheimer’s disease treatments.