Abstract
HAMP domains, ∼55 amino acid motifs first identified in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases, operate as signal mediators in two-component signal transduction proteins. A bioinformatics study identified a coevolving signal-accepting network of 10 amino acids in membrane-delimited HAMP proteins. To probe the functionality of this network we used a HAMP containing mycobacterial adenylyl cyclase, Rv3645, as a reporter enzyme in which the membrane anchor was substituted by the Escherichia coli chemotaxis receptor for serine (Tsr receptor) and the HAMP domain alternately with that from the protein Af1503 of the archaeon Archaeoglobus fulgidus or the Tsr receptor. In a construct with the Tsr-HAMP, cyclase activity was inhibited by serine, whereas in a construct with the HAMP domain from A. fulgidus, enzyme activity was not responsive to serine. Amino acids of the signal-accepting network were mutually swapped between both HAMP domains, and serine signaling was examined. The data biochemically tentatively established the functionality of the signal-accepting network. Based on a two-state gearbox model of rotation in HAMP domain-mediated signal propagation, we characterized the interaction between permanent and transient core residues in a coiled coil HAMP structure. The data are compatible with HAMP rotation in signal propagation but do not exclude alternative models for HAMP signaling. Finally, we present data indicating that the connector, which links the α-helices of HAMP domains, plays an important structural role in HAMP function.
Highlights
HAMP domains accept signals from membrane receptors and propagate them possibly via rotation
To probe the functionality of this network we used a HAMP containing mycobacterial adenylyl cyclase, Rv3645, as a reporter enzyme in which the membrane anchor was substituted by the Escherichia coli chemotaxis receptor for serine (Tsr receptor) and the HAMP domain alternately with that from the protein Af1503 of the archaeon Archaeoglobus fulgidus or the the serine (Tsr) receptor
A Coevolving Network within HAMP as Signal Receiver—A bioinformatics study showed that in HAMP domains, key positions evolved in three defined networks (4) within which residues are thought to act in a concerted way to yield a specific functionality
Summary
HAMP domains accept signals from membrane receptors and propagate them possibly via rotation. Histidine kinases and chemoreceptors typically show an extracytoplasmic sensor domain, a transmembrane segment, and a cytoplasmic output or transmitter domain Such ensembles are found in bacterial membrane-anchored adenylyl cyclases, Ser/Thr protein kinases, and phosphatases (2). These sensor proteins often possess distinct regions between extra- and intracellular domains that presumably undergo conformational changes during signal propagation One of these regions is the HAMP domain, first found in histidine kinases, adenylyl cyclases (ACs), methyl-accepting chemotaxis proteins, and phosphatases (3) and present in many other proteins (4). Serine regulation was achieved by substitution of Ala291 with Phe, supporting the notion of rotation as a potentially common mechanism in HAMP-mediated signal propagation (16) We used this Tsr-AC reporter system with the Tsr or Af1503 HAMP domains and carried out a targeted mutational analysis based on sequence and structural features to investigate the role of particular amino acids in signal propagation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
