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UBC Theses and Dissertations

Dissecting the biophysical mechanisms of phase separation by the myobacterium tuberculosis ABC transporter rv1747 Hui, Lok Tin


The ABC transporter Rv1747, which is important for Mycobacterium tuberculosis (Mtb) growth in infected host cells, has a cytoplasmic regulatory module with two phosphothreonine-binding FHA domains joined by a flexible linker with multiple phospho-acceptor threonines (pThr). In vitro, the isolated regulatory module, Rv1747¹⁻³¹⁰, undergoes phosphorylation-enhanced phase separation upon linker phosphorylation by Mtb serine/threonine kinases. Phosphorylation-based phase separation was proposed to result from multivalent intra/intermolecular FHA-pThr interactions. To further dissect the mechanisms of Rv1747 phase separation, I have investigated the factors that influence droplet formation. Visualized with a temperature-controlled microscope, I found that warming phosphorylated Rv1747¹⁻³¹⁰ droplets from 22 oC to 37 oC modestly reduced their size. Studies with Rv1747 truncation fragments and mutants demonstrated that the FHA-1 domain has a predominant role in phosphorylation-enhanced phase separation. Substitution of a key binding residue, S47, in the FHA-1 domain to alanine impaired droplet formation. In contrast, variants with the analogous mutation, S248A, in the FHA-2 domain, or lacking the FHA-2 domain entirely, still underwent phase separation. However, the resulting droplets were gel-like, indicating both FHA domains are required for “regular” droplet formation. The role of the intrinsically disordered linker was also studied. A polypeptide corresponding to the isolated linker did not phase separate. Also, a comparison of Rv1747¹⁻²¹³ (FHA-1 domain and linker) versus Rv1747¹⁻¹¹⁰ (FHA-1 domain only) by NMR spectroscopy indicated the linker residues do not interact with the FHA-1 domain. Substitution of threonine residues in the linker to alanines revealed that T152 is a key residue for the phosphorylation-enhanced phase separation of the regulatory module. I also found that Rv1747¹⁻³¹⁰ treated with PknF in the absence of ATP still underwent phase separation. Based on studies using inactive PknF variants, I hypothesize that PknF with activating pThr residues directly contributes to droplet formation by bridging the FHA domains of Rv1747¹⁻³¹⁰. Lastly, I observed that protein partnerships with the stress protein Rv2623, and Rv1747 linker phosphopeptides influenced Rv1747¹⁻³¹⁰ droplet formation. Although the underlying mechanisms have not been fully established, my studies support the notion that Rv1747 can be controlled by cellular pathways impacting the phase separation of its regulatory module.

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