CYSTEINE AND DISULFIDE SCANNING REVEALS A REGULATORY ALPHA-HELIX IN THE CYTOPLASMIC DOMAIN OF THE ASPARTATE RECEPTOR

The transmembrane, homodimeric aspartate receptor of Escherichia coil and Salmonella typhimurium controls the chemotactic response to aspart ate, an attractant, by regulating the activity of a cytoplasmic histid ine kinase, The cytoplasmic domain of the receptor plays a central rol e in both kinase regulation and sensory adaptation, although its struc ture and regulatory mechanisms are unknown, The present study utilizes cysteine and disulfide scanning to probe residues Leu-250 through Gln -309, a region that contains the first of two adaptive methylation seg ments within the cytoplasmic domain, Following the introduction of con secutive cysteine residues by scanning mutagenesis, the measurement of sulfhydryl chemical reactivities reveals an alpha-helical pattern of exposed and buried positions spanning residues 270-309. This detected helix, termed the ''first methylation helix,'' is strongly amphiphilic ; its exposed face is highly anionic and possesses three methylation s ites, while its buried face is hydrophobic, In vivo and in vitro assay s of receptor function indicate that inhibitory cysteine substitutions are most prevalent on the buried face of the first methylation helix, demonstrating that this face is involved in a critical packing intera ction. The buried face is further analyzed by disulfide scanning which reveals three ''lock-on'' disulfides that covalently trap the recepto r in its kinase-activating state, Each of the lock-on disulfides cross links the buried faces of the two symmetric first methylation helices of the dimer, placing these helices in direct contact at the subunit i nterface, Comparative sequence analysis of 56 related receptors sugges ts that the identified helix is a conserved feature of this large rece ptor family, wherein it is likely to play a general role in adaptation and kinase regulation, Interestingly, the rapid rates and promiscuous nature of disulfide formation reactions within the scanned region rev eal that the cytoplasmic domain of the full-length, membrane-bound rec eptor has a highly dynamic structure. Overall, the results demonstrate that cysteine and disulfide scanning can identify secondary structure elements and functionally important packing interfaces, even in prote ins that are inaccessible to other structural methods.