UC Berkeley

The survival of the pathogenic bacterium, Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, depends on its ability to sense and respond to a hostile and constantly changing environment within the host. Cell-wall components--such as peptidoglycan, arabinogalactan, mycolic acids and lipoarabinomannan--perform critical structural and biological functions that allow Mtb to survive and persist inside of human cells. Despite the critical roles of the cell wall in the pathogenicity of Mtb, surprisingly little is known about how this complex structure is built and remodeled in response to environmental cues. Recent evidence, however, implicates membrane-bound serine/threonine protein kinases (STPKs) in the coordinating cell-wall synthesis with growth and division. These eukaryotic-like STPKs play critical roles in Mtb signal transduction by sensing extracellular stimuli and catalyzing the reversible phosphorylation of cytoplasmic target proteins.

To further understand the mechanisms that regulate STPKs and cell wall architecture in Mtb, I biochemically and structurally characterized the extracellular sensor domain of one STPK receptor kinase, PknH. Initial analysis of recombinantly produced PknH sensor domain by size-exclusion chromatography and small-angle x-ray scattering indicated the protein was soluble but completely unfolded. On-column oxidative refolding produced a properly folded protein that crystallized as a monomer with a novel protein fold made up of six alpha helices, seven beta strands, and two disulfide bonds. The PknH sensor domain has a large conserved cleft with a mixed polar and hydrophobic surface. These results indicated PknH binds a small molecule ligand which may affect its quarternary structure and/or its localization.

In order to determine what ligand binds to the PknH sensor domain, I conducted native gel binding assays using purified Mtb arabinogalactan, lipoarabinomannan, and lipomannan. These assays indicated that in vitro, the PknH sensor domain does not bind to any of these purified cell wall components. Affinity chromatography with His₆-tagged PknH incubated with Mtb H37Rv cell lysate followed by intact mass, LC-MS, and ESI-MS analysis indicated that PknH does not bind glycolipids such as phosphoinositol or Ac₁PIM₂. Although the ligand for PknH was not identified, these studies set the stage for future researchers to identify the ligand and discover the function of PknH.