A protein design- and structure-guided interrogation of signal transduction mechanisms

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Project Summary/Abstract Living cells have evolved intricate mechanisms to detect their environment and transduce signals across biological membranes, inducing responses in organismal behavior. Despite the prevalence of these receptors, our understanding of the discrete mechanisms by which signals are propagated across membranes is still evolving. In this area, histidine kinases (HKs) are a predominant class of membrane receptors in bacteria, fungi, and plants that regulate growth, survival, or pathogenicity. HKs sense diverse extracellular stimuli and transduce a signal across the membrane and through multiple subdomains, activating a phosphorylation cascade and inducing a transcriptional response. Early models proposed that HKs do so through large, rigid body shifts after sensing extracellular stimuli. Subsequent work indicates that signals are passed between HK subdomains in a step-wise manner, often through changes in protein dynamics, informing the hypothesis that signal transduction is the result of thermodynamic coupling between subdomains of the HK complex. This further implies that many conformations may be adopted in the course of HK signaling. To investigate the molecular and biophysical basis of HK specificity and signal transduction, we propose a structure and protein design approach to interrogate energetic thresholds, sensor specificity, and conformational bias in transmembrane signaling. First, rational and de novo design will be used to generate non-native, thermodynamically tunable sensor domains to determine what ligand-induced energetic response is sufficient to initiate signaling. This will be complemented with experimental characterization of synthetic, orthogonal sensor domains identified through a sequence- and structure-guided neural network algorithm. In parallel, we will pursue X-ray crystallography and cryo-electron microscopy to elucidate the structure of HK complexes or isolated subdomains in various signaling states, to inform assembly of structure-conformation-function relationships. This research will significantly advance our understanding of energetics and dynamics in transmembrane signal transduction while advancing our ability to use protein design and computational biology to interrogate and engineer complex biophysical mechanisms. The proposed efforts will also directly fulfill the training goals of my postdoctoral tenure, affording the necessary skills to prepare me for an independent research career studying and engineering signal transduction mechanisms.