A family of related transmembrane chemoreceptors mediates chemotaxis in Escherichia coli, Salmonella typhimurium, and many other bacteria (1-3). These integral membrane proteins are homodimers (5) organized as diagrammed in Fig. 1. The structure of the periplasmic domain, determined by x-ray crystallography (6), is two four-helix bundles, one from each monomer, that interact along a central axis. The two central helices (at and a4) of each bundle are >70 A long and extend to the membrane (Fig. 1A). The organization of the transmembrane domain, deduced from patterns of disulfide formation between introduced cysteines (4, 7) is a bundle of four helical segments (Fig. 1B) that appear to be uninterrupted extensions of the four central helices of the periplasmic domain (4, 6-9). The cytoplasmic domain interacts with a kinase and an accessory protein to form a ternary complex that controls the rotational bias of flagellar motors by phosphorylation of a response regulator (10, 11). Sensory adaptation is mediated by methylation of specific glutamyl residues in the cytoplasmic domain of the chemoreceptor (1-3).How does ligand binding to the periplasmic domain of a chemoreceptor cause a change in the cytoplasmic domain that alters the phosphorylation activity of the ternary complex and ultimately cellular behavior? Several lines of evidence (5,8,9,(12)(13)(14) indicate that transmembrane signaling in the chemoreceptors involves a conformational change within the stable dimeric protein. In this study we addressed the importance for transmembrane signaling of movement between specific pairs of transmembrane helices. We used sulfhydryl cross-linking to constrain movement between helices in cysteine-containing forms of the chemoreceptor Trg and assessed the ability of the constrained receptors to mediate tactic responses in intact cells. MATERIALS AND METHODSStrains and Plasmids. Derivatives of pGB1 (15), which carries trg under the control of the tac promoter, lacIq and bla, were introduced into CP177, a strain of E. coli K-12 deleted for trg but otherwise wild type for chemotaxis and motility (16). The plasmids carried an altered trg coding for a product devoid of cysteine (Trg-C23S) or derivatives of that gene coding for proteins with a cysteine introduced at one or two specific Abbreviations: CCW, counterclockwise; CW, clockwise.

We applied mutational analysis to a protein domain that functions in neither catalysis nor binding but, rather, in transmembrane signaling. The domain is part of chemoreceptor Trg from Escherichia coli. It contains four transmembrane segments, two from each subunit of the homodimer. We used cysteine scanning to investigate the functional importance of each of54 residues in the two transmembrane segments. Cysteines at some positions resulted in subtle but significant reductions in tactic response. Those positions defined a specific helical face on each segment, implying that the segments function as helices. The functionally important faces corresponded to structural, helical packing faces identified independently by biochemical studies. All functionally impaired receptors exhibited altered signaling properties, either reduced signaling upon stimulation or induced signaling in the absence of stimulation. The distribution of substitutions creating these two phenotypes implied that conformational signaling involves movement between the two transmembrane helices within a subunit and that signaling is optimal when stable interactions are maintained across the interface between subunits.Four transmembrane receptors mediate chemotaxis by Escherichia coli (1-3). Recognition of positive stimuli generates counterclockwise (CCW) rotation of bacterial flagella, which in turn results in coordinated swimming. Recognition of negative stimuli generates clockwise (CW) rotation and uncoordinated tumbles. Chemoreceptor Trg recognizes sugaroccupied galactose-and ribose-binding proteins and thereby mediates chemotactic response to those sugars. Chemoreceptors are homodimers (4-6). Receptor monomers, "60 kDa, have a transmembrane domain consisting of two hydrophobic segments, a periplasmic, ligand-binding domain and a highly conserved cytoplasmic domain that mediates intracellular signaling by controlling a noncovalently associated protein kinase and that mediates adaptation through dynamic methylation and demethylation of several glutamyl residues. An understanding of transmembrane signaling in chemoreceptors of E. coli should be relevant not only to methyl-accepting taxis proteins in other bacteria (7) but also to the many related environmental sensor proteins in prokaryotes (1, 3) and eukaryotes (8).The monomer-dimer equilibrium for chemoreceptors strongly favors dimers and does not appear to shift in the course of the sensory cycle (4), implying that transmembrane signaling involves conformational changes within the dimer. It is not known what features of the domain are important for or involved in transmembrane signaling. We approached these issues with mutational analysis by "cysteine scanning." The approach has many of the features and advantages of "alaninescanning mutagenesis" (9). Cysteine is an attractive choice for scanning mutagenesis of transmembrane segments because itsThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisemen...

An assay of GDP-fucose:polypeptide fucosyltransferase has been established. The enzyme catalyzes the reaction that attaches fucose through an O-glycosidic linkage to a conserved serine or threonine residue in EGF domains. The assay uses recombinant human factor VII EGF-1 domain as acceptor substrate and GDP-fucose as donor substrate. Synthetic peptides with sequences taken from five proteins previously shown to contain O-linked fucose (Harris and Spellman, 1993; Glycobiology, 3, 219-224) did not serve as efficient acceptor substrates. These synthetic peptides did not compromise complete EGF domains and did not contain all six cysteine residues that define the EGF structure. Therefore, the enzyme appears to require more than just a consensus primary sequence and likely requires that the EGF domain disulfide bonds be properly formed. The enzymatic reaction showed linear dependency of its activity on time, amount of enzyme, and substrates. Although the enzyme did not exhibit an absolute requirement for Mn2+, enzymatic activity did increase ten fold in the presence of 50 mM MnCl2. The in vitro glycosylation reaction resulted in complete conversion of the acceptor substrate to glycosylated product, and characterization of the purified product by electrospray mass spectrometry revealed that one fucose was added onto the polypeptide. Most of the enzymatic activity was found to be in the soluble fraction of CHO cell homogenates. However, when enzyme was prepared from rat liver in the presence of protease inhibitors, 37% of the activity was recovered by Triton X-100 extraction of the membrane particles after extensive aqueous washes. The result suggests that the enzyme is probably a membrane protein and, by analogy with other glycosyltransferases, probably has a 'stem' region that is very susceptible to proteolysis.

The first epidermal growth factor-like domain (EGF-1) from blood coagulation factor VII (FVII) contains two unusual O-linked glycosylation sites at Ser-52 and Ser-60. We report here a detailed study of the effect of O-fucosylation at Ser-60 on the structure of FVII EGF-1, its Ca2+-binding affinity, and its interaction with tissue factor (TF). The in vitro fucosylation of the nonglycosylated FVII EGF-1 was achieved by using O-fucosyltransferase purified from Chinese hamster ovary cells. Distance and dihedral constraints derived from NMR data were used to determine the solution structures of both nonglycosylated and fucosylated FVII EGF-1 in the presence of CaCl2. The overall structure of fucosylated FVII EGF-1 is very similar to the nonfucosylated form even for the residues near the fucosylation site. The Ca2+ dissociation constants (Kd) for the nonfucosylated and fucosylated FVII EGF-1 were found to be 16.4 +/- 1.8 and 8.6 +/- 1.4 mM, respectively. The FVII EGF-1 domain binds to the extracellular part of TF with a low affinity (Kd approximately 0. 6 mM), and the addition of fucose appears to have no effect on this affinity. These results indicate that the FVII EGF-1 alone cannot form a tight complex with TF and suggest that the high binding affinity of FVIIa for TF requires cooperative interaction among the four domains in FVII with TF. Although the fucose has no significant effect on the interaction between TF and the individual FVII EGF-1 domain, it may affect the interaction of full-length FVIIa with TF by influencing its Ca2+-binding affinity.

The transmembrane domain of chemoreceptor Trg from Escherichia coli contains four transmembrane segments in its native homodimer, two from each subunit. We had previously used mutational analysis and sulfhydryl crosslinking between introduced cysteines to obtain data relevant to the three-dimensional organization of this domain. In the current study we used Fourier analysis to assess these data quantitatively for periodicity along the sequences of the segments. The analyses provided a strong indication of a-helical periodicity in the first transmembrane segment and a substantial indication of that periodicity for the second segment. On this basis, we considered both segments as idealized a-helices and proceeded to model the transmembrane domain as a unit of four helices. For this modeling, we calculated helical crosslinking moments, parameters analogous to helical hydrophobic moments, as a quantitative way of condensing and utilizing a large body of crosslinking data. Crosslinking moments were used to define the relative separation and orientation of helical pairs, thus creating a quantitatively derived model for the transmembrane domain of Trg. Utilization of Fourier transforms to provide a quantitative indication of periodicity in data from analyses of transmembrane segments, in combination with helical crosslinking moments to position helical pairs should be useful in modeling other transmembrane domains.