Biomedical subjects

Mingshan Li

Publications and source records attributed to Mingshan Li.

4 recordsLinked to original sources

PubMed2006

Nanodiscs separate chemoreceptor oligomeric states and reveal their signaling properties.

Bacterial chemoreceptors are transmembrane homodimers that can form trimers, higher order arrays, and extended clusters as part of signaling complexes. Interactions of dimers in oligomers are thought to confer cooperativity and cross-receptor influences as well as a 35-fold gain between ligand binding and altered kinase activity. In addition, higher order interactions among dimers are necessary for the observed patterns of assistance in adaptational modification among different receptors. Elucidating mechanisms underlying these properties will require defining which receptor functions can be performed by dimers and which require specific higher order interactions. However, such an assignment has not been possible. Here, we used Nanodiscs, an emerging technology for manipulating membrane proteins, to prepare small particles of lipid bilayer containing one or only a few chemoreceptor dimers. We found that receptor dimers isolated in individual Nanodiscs were readily modified, bound ligand, and performed transmembrane signaling. However, they were hardly able to activate the chemotaxis histidine kinase. Instead, maximal activation and thus full-range control of kinase occurred preferentially in discs containing approximately three chemoreceptor dimers. The sharp dependence of kinase activation on this number of receptors per dimer implies that the core structural unit of kinase activation and control is a trimer of dimers. Thus, our observations demonstrate that chemoreceptor transmembrane signaling does not require oligomeric organization beyond homodimers and implicate a trimer of dimers as the unit of downstream signaling.

Dimerization↗

PubMed2006

The carboxyl-terminal linker is important for chemoreceptor function.

Sensory adaptation in bacterial chemotaxis is mediated by chemoreceptor methylation and demethylation. In Escherichia coli, methyltransferase CheR and methylesterase CheB bind both substrate sites and a carboxyl-terminal pentapeptide sequence carried by certain receptors. Pentapeptide binding enhances enzyme action, an enhancement required for effective adaptation and chemotaxis. Pentapeptides are linked to the conserved body of chemoreceptors through a notably variable sequence of 30-35 residues. We created nested deletions from the distal end of this linker in chemoreceptor Tar. Chemotaxis was eliminated by deletion of 20-40 residues and reduced by shorter deletions. This did not reflect generalized disruption, because all but the most extremely truncated receptors activated kinase, were substrates for adaptational modification and performed transmembrane signalling. In contrast, linker truncations reduced rates of adaptational modification in parallel with chemotaxis. We concluded the linker is important for chemotaxis because of its role in adaptational modification. Effects of linker truncations on CheR binding to receptor-borne pentapeptide implied linker (i) makes pentapeptide available to modification enzymes by separation from the helical receptor body, and (ii) is a flexible arm allowing dual binding of enzyme to pentapeptide and modification site. The data suggest linker and the helix from which it emerges are structurally dynamic.

Amino Acid Sequence↗

PubMed2005

Adaptational assistance in clusters of bacterial chemoreceptors.

Sensory adaptation of low-abundance chemoreceptors in Escherichia coli requires assistance from high-abundance receptors, because only high-abundance receptors carry the carboxyl-terminal pentapeptide sequence NWETF that enhances adaptational covalent modification. Using membrane vesicles containing both high-abundance receptor Tar and low-abundance receptor Trg, we observed effective assistance in vitro for all three adaptational modifications: methylation, demethylation and deamidation. These results demonstrated that adaptational assistance involves not only the previously documented assistance for methylation but also assistance for the two CheB-catalysed reactions. We determined rates of assisted methylation and demethylation at many ratios of assisting to assisted receptor. Analysis by a model of assistance indicated one Tar dimer could assist seven Trg dimers in methylation or five in demethylation, defining assistance neighbourhoods. These neighbourhoods were larger than a trimer of homodimers, required only receptors and were minimally affected by formation of signalling complexes. Time courses of assisted Trg methylation in membranes with low amounts of Tar showed that assisting receptors did not diffuse beyond initial neighbourhoods for at least two hours. Taken together, these observations indicate that chemoreceptors can form stable neighbourhoods larger than trimers in the absence of other chemotaxis proteins. Such interactions are likely to occur in natural receptor clusters in vivo.

Adaptation, Physiological↗

PubMed2004

Cellular stoichiometry of the components of the chemotaxis signaling complex.

The chemotactic sensory system of Escherichia coli comprises membrane-embedded chemoreceptors and six soluble chemotaxis (Che) proteins. These components form signaling complexes that mediate sensory excitation and adaptation. Previous determinations of cellular content of individual components provided differing and apparently conflicting values. We used quantitative immunoblotting to perform comprehensive determinations of cellular amounts of all components in two E. coli strains considered wild type for chemotaxis, grown in rich and minimal media. Cellular amounts varied up to 10-fold, but ratios between proteins varied no more than 30%. Thus, cellular stoichiometries were almost constant as amounts varied substantially. Calculations using those cellular stoichiometries and values for in vivo proportions of core components in complexes yielded an in vivo stoichiometry for core complexes of 3.4 receptor dimers and 1.6 CheW monomers for each CheA dimer and 2.4 CheY, 0.5 CheZ dimers, 0.08 CheB, and 0.05 CheR per complex. The values suggest a core unit of a trimer of chemoreceptor dimers, a dimer (or two monomers) of kinase CheA, and two CheW. These components may interact in extended arrays and, thus, stoichiometries could be nonintegral. In any case, cellular stoichiometries indicate that CheY could be bound to all signaling complexes and this binding would recruit essentially the entire cellular complement of unphosphorylated CheY, and also that phosphatase CheZ, methylesterase CheB, and methyltransferase CheR would be present at 1 per 2, per 14, and per 20 core complexes, respectively. These characteristic ratios will be important in quantitative treatments of chemotaxis, both experimental and theoretical.

Bacterial Proteins↗