title: Structure-function relationships in the HAMP and proximal signaling domains of the aerotaxis receptor Aer.
abstract: Aer, the Escherichia coli aerotaxis receptor, faces the cytoplasm, where the PAS (Per-ARNT-Sim)-flavin adenine dinucleotide (FAD) domain senses redox changes in the electron transport system or cytoplasm. PAS-FAD interacts with a HAMP (histidine kinase, adenylyl cyclase, methyl-accepting protein, and phosphatase) domain to form an input-output module for Aer signaling. In this study, the structure of the Aer HAMP and proximal signaling domains was probed to elucidate structure-function relationships important for signaling. Aer residues 210 to 290 were individually replaced with cysteine and then cross-linked in vivo. The results confirmed that the Aer HAMP domain is composed of two alpha-helices separated by a structured loop. The proximal signaling domain consisted of two alpha-helices separated by a short undetermined structure. The Af1503 HAMP domain from Archaeoglobus fulgidus was recently shown to be a four-helix bundle. To test whether the Af1503 HAMP domain is a prototype for the Aer HAMP domain, the latter was modeled using coordinates from Af1503. Several findings supported the hypothesis that Aer has a four-helix HAMP structure: (i) cross-linking independently identified the same residues at the dimer interface that were predicted by the model, (ii) the rate of cross-linking for residue pairs was inversely proportional to the beta-carbon distances measured on the model, and (iii) clockwise lesions that were not contiguous in the linear Aer sequence were clustered in one region in the folded HAMP model, defining a potential site of PAS-HAMP interaction during signaling. In silico modeling of mutant Aer proteins indicated that the four-helix HAMP structure was important for Aer stability or maturation. The significance of the HAMP and proximal signaling domain structure for signal transduction is discussed.
authors.affiliation: Division of Microbiology and Molecular Genetics, Loma Linda University, Loma Linda, CA 92350, USA.
authors.forename: Kylie J
authors.initials: KJ
authors.lastname: Watts
authors.forename: Mark S
authors.initials: MS
authors.lastname: Johnson
authors.forename: Barry L
authors.initials: BL
authors.lastname: Taylor
mesh_terms: D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins; D008954:Models, Biological; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D013329:Structure-Activity Relationship
chemical_list: C108679:Aer protein, E coli; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D036341:Intercellular Signaling Peptides and Proteins; D003545:Cysteine