Role of Signal Transduction Domains in Histidine Kinase Evolution and Activity
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Role of Signal Transduction Domains in Histidine Kinase Evolution and Activity

  • Author(s): Mensa, Bruk
  • Advisor(s): DeGrado, William F
  • et al.
Abstract

The process by which various upstream sensor and signal-transduction domains of bacterial histidine kinases (HKs) modulate the activity of the conserved autokinase domain remains poorly understood. Specifically, why do most HKs contain modularly inserted signal transduction domains? How do HKs robustly evolve and finetune the coupling between stimulus sensor domains and the conserved autokinase domain, which are often separated by 10s of nanometers? What is the role of these intervening domains in fine-tuning signaling parameters such as the minimum/maximum responsiveness, mid-point, and steepness of signal transition of an HK? In this work, we examine signal transduction through model E. coli HKs, PhoQ and CpxA, which contain one of the most abundant signal transduction domains in HKs, the HAMP domain. We first generate a large set of single-point mutants of PhoQ, and simultaneously measure the signaling state of the ligand-binding sensor and the kinase activity of the autokinase in vitro, at several inducing ligand concentrations to assess the coupling between these two domains. We demonstrate that point mutants in the HAMP signal transduction domain significantly modulate the coupled behavior of the sensor and autokinase, producing markedly varied ligand-dependent responses. We further use the insertion of poly-glycine motifs (Gly7) to decouple domains from one another and qualitatively show that, intrinsically, the sensor domain has a drastically poor ligand-dependent state transition propensity, and similarly, the autokinase domain has a drastically high basal kinase activity. The HAMP domain strongly couples to both domains and is sufficient to adjust these propensities to what is observed in the full length PhoQ. We suggest that signal transduction in PhoQ occurs by an allosteric coupling mechanism, in which the HAMP domain strongly couples to and acts in opposition the underlying signaling state equilibria of PhoQ such that it is maximally responsive to physiologically relevant ranges of stimuli. We demonstrate the same phenomenon in two other E. coli HAMP containing HKs, CpxA and BaeS, and suggest this may be a common theme in the evolution of signal transduction domains in HKs. In order to quantitatively examine the feasibility of modulating various ligand-dependent properties that inform HK function through evolution, we next establish and experimentally fit a three-domain, two-state equilibrium allosteric signaling model. We demonstrate that small changes to the HAMP domain sequence allow for robust modulation of the signaling ensemble and provide quantitative measures for the strong modulation of both sensor and autokinase domains by the HAMP, as well as the effects of point-mutations and Gly7 insertions. We more fully examine the ability of the HAMP to couple strongly and influence the sensor and autokinase domains of PhoQ by introducing a large library of variants in the HAMP four-helix bundle hydrophobic core, as well as the junction between the HAMP and autokinase domains (the S-Helix) and selecting for variants with high PhoQ activity. We find that destabilizing the HAMP four-helix bundle hydrophobic core does indeed lead to higher kinase activity. Furthermore, we find that the wild-type S-Helix sequence is enriched in the high-activity population, along with sequences with comparable polarity or poor helical propensity. Taken together, these observations lend credence to the hypothesis that the thermodynamically preferred signaling state of the HAMP behaves as a negative allosteric regulator of the autokinase, and that this regulation is alleviated by destabilizing the core helical bundle structure as well as the alpha-helical motif that connects it to the autokinase. We investigate this relationship further using a deep learning method to establish sequence-activity predictive relationships and extract structural features that are essential for this behavior. Finally, we examine the question of whether the HAMP domain exists in two distinct structural states, or rather conformational ensembles that can be classified into one of two functional states. We examine signaling through the HAMP domain of an E. coli histidine kinase, CpxA, by constructing a small library of structurally diverse inputs into the HAMP domain and evaluate the resulting autokinase activity as a function of several S-helix point mutations. This analysis allows us to discern the relationship between different signal inputs into the HAMP domain as the linkage to the output domain (autokinase) is varied. We find that the HAMP seems to have a multiconformational landscape that is not explained by 2 unique structural conformations. In this thesis, we show that the insertion of signal transduction domains in HKs can significantly alter both the intrinsic behaviors of sensor and autokinase domains, as well as the coupling between them. These properties can be well-described through a coupled two-state allosteric mechanism, and easily finetuned through simple mutations to the signal transduction domain and its linkage to adjacent domains to achieve the desired physiologically relevant activity profile.

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