10 research outputs found
New Insights into Bacterial Chemoreceptor Array Structure and Assembly from Electron Cryotomography
Bacterial chemoreceptors cluster in highly ordered, cooperative, extended arrays with a conserved architecture, but the principles that govern array assembly remain unclear. Here we show images of cellular arrays as well as selected chemoreceptor complexes reconstituted in vitro that reveal new principles of array structure and assembly. First, in every case, receptors clustered in a trimers-of-dimers configuration, suggesting this is a highly favored fundamental building block. Second, these trimers-of-receptor dimers exhibited great versatility in the kinds of contacts they formed with each other and with other components of the signaling pathway, although only one architectural type occurred in native arrays. Third, the membrane, while it likely accelerates the formation of arrays, was neither necessary nor sufficient for lattice formation. Molecular crowding substituted for the stabilizing effect of the membrane and allowed cytoplasmic receptor fragments to form sandwiched lattices that strongly resemble the cytoplasmic chemoreceptor arrays found in some bacterial species. Finally, the effective determinant of array structure seemed to be CheA and CheW, which formed a âsuperlatticeâ of alternating CheA-filled and CheA-empty rings that linked receptor trimers-of-dimer units into their native hexagonal lattice. While concomitant overexpression of receptors, CheA, and CheW yielded arrays with native spacing, the CheA occupancy was lower and less ordered, suggesting that temporal and spatial coordination of gene expression driven by a single transcription factor may be vital for full order, or that array overgrowth may trigger a disassembly process. The results described here provide new insights into the assembly intermediates and assembly mechanism of this massive macromolecular complex
Potassium-selective block of barium permeation through single KcsA channels
Ba2+, a doubly charged analogue of K+, specifically blocks K+ channels by virtue of electrostatic stabilization in the permeation pathway. Ba2+ block is used here as a tool to determine the equilibrium binding affinity for various monovalent cations at specific sites in the selectivity filter of a noninactivating mutant of KcsA. At high concentrations of external K+, the block-time distribution is double exponential, marking at least two Ba2+ sites in the selectivity filter, in accord with a Ba2+-containing crystal structure of KcsA. By analyzing block as a function of extracellular K+, we determined the equilibrium dissociation constant of K+ and of other monovalent cations at an extracellular site, presumably S1, to arrive at a selectivity sequence for binding at this site: Rb+ (3 ÂľM) > Cs+ (23 ÂľM) > K+ (29 ÂľM) > NH4+ (440 ÂľM) >> Na+ and Li+ (>1 M). This represents an unusually high selectivity for K+ over Na+, with |ÎÎG0| of at least 7 kcal molâ1. These results fit well with other kinetic measurements of selectivity as well as with the many crystal structures of KcsA in various ionic conditions
Defining a Key ReceptorâCheA Kinase Contact and Elucidating Its Function in the Membrane-Bound Bacterial Chemosensory Array: A Disulfide Mapping and TAM-IDS Study
The
three core components of the ubiquitous bacterial chemosensory
array î¸ the transmembrane chemoreceptor, the histidine kinase
CheA, and the adaptor protein CheW î¸ assemble to form a membrane-bound,
hexagonal lattice in which receptor transmembrane signals regulate
kinase activity. Both the regulatory domain of the kinase and the
adaptor protein bind to overlapping sites on the cytoplasmic tip of
the receptor (termed the protein interaction region). Notably, the
kinase regulatory domain and the adaptor protein share the same fold
constructed of two SH3-like domains. The present study focuses on
the structural interface between the receptor and the kinase regulatory
domain. Two models have been proposed for this interface: Model 1
is based on the crystal structure of a homologous Thermotoga complex
between a receptor fragment and the CheW adaptor protein. This model
has been used in current models of chemosensory array architecture
to build the receptorâCheA kinase interface. Model 2 is based
on a newly determined crystal structure of a homologous Thermotoga
complex between a receptor fragment and the CheA kinase regulatory
domain. Both models present unique strengths and weaknesses, and current
evidence is unable to resolve which model best describes contacts
in the native chemosensory arrays of <i>Escherichia coli</i>, <i>Salmonella typhimurium</i>, and other bacteria. Here
we employ disulfide mapping and tryptophan and alanine mutation to
identify docking sites (TAM-IDS) to test Models 1 and 2 in well-characterized
membrane-bound arrays formed from <i>E. coli</i> and <i>S. typhimurium</i> components. The results reveal that the native
array interface between the receptor protein interaction region and
the kinase regulatory domain is accurately described by Model 2, but
not by Model 1. In addition, the results show that the interface possesses
both a structural function that contributes to stable CheA kinase
binding in the array and a regulatory function central to transmission
of the activation signal from receptor to CheA kinase. Onâoff
switching alters the disulfide formation rates of specific Cys pairs
at the interface, but not most Cys pairs, indicating that signaling
perturbs localized regions of the interface. The findings suggest
a simple model for the rearrangement of the interface triggered by
the attractant signal and for longer range transmission of the signal
in the chemosensory array