HAMP Domain-mediated Signal Transduction Probed with a Mycobacterial Adenylyl Cyclase as a Reporter

Abstract

Background: HAMP domains accept signals from membrane receptors and propagate them possibly via rotation. Results: Using targeted mutations based on bioinformatics, sequence comparisons, and structures, we characterize the role of particular amino acids in signaling. Conclusion: Results are compatible with a gearbox model of HAMP rotation. Significance: Various models of HAMP domain-mediated signaling are testable.

The S-Helix Determines the Signal in a Tsr Receptor/Adenylyl Cyclase Reporter

“…the receptor signal was reverted, in stark contrast to all past chimeras ( Fig. 1, bottom) (11,16,20). This indicated that the linker sequence between HAMP and cyclase domains might be critical for cytoplasmic signal processing.…”

“…This indicated that the origin of the HAMP domain and/or the type of connection between HAMP and AC might be important for signal propagation. In these former studies we mainly used the membrane-bound mycobacterial AC Rv3645 for generation of functional chimeras with the Tsr receptor domain and demonstrated that all chimeras were inhibited by serine in a concentration-dependent fashion (11,16). A comparison of the linker sequences between the respective HAMP and catalytic domains from the ACs from A. maxima (CyaG) and M. tuberculosis (Rv3645) identified a 25-residue segment in CyaG that is absent in the AC Rv3645 (Fig.…”

“…The tripartite domain organization of HAMP-containing bacterial ACs resembles bacterial chemotaxis proteins (methyl-accepting proteins of chemotaxis) such as Tsr for serine from E. coli (40). Functional chimeras between these methyl-accepting proteins of chemotaxis and ACs have been generated (11,16,20).…”

“…In many signal transduction systems, HAMP 2 domains (histidine kinase, adenylyl cyclase, methyl-accepting proteins of chemotaxis, protein-phosphatases (17)), which are four-bundle parallel coiled coils of about 55 residues, are interspaced between sensory and output domains (18 -19). We have studied HAMP-mediated signal transduction across the cytoplasmic membrane in vitro and in vivo using chimeras composed of the E. coli chemotaxis receptor for serine (Tsr) as a sensor, different HAMP domains, and the AC catalytic domains CyaG from Arthrospira platensis and Rv3645 from Mycobacterium tuberculosis (11,16,18,20). In these constructs, serine inhibited AC activity in vitro and in vivo (11,20).…”

“…In several instances it has been demonstrated that between one-and two-component signaling systems individual domains are interchangeable without loss of function (11)(12)(13)(14)(15)(16). In many signal transduction systems, HAMP 2 domains (histidine kinase, adenylyl cyclase, methyl-accepting proteins of chemotaxis, protein-phosphatases (17)), which are four-bundle parallel coiled coils of about 55 residues, are interspaced between sensory and output domains (18 -19).…”