Defining a Key Receptor–CheA
Kinase Contact
and Elucidating Its Function in the Membrane-Bound Bacterial Chemosensory
Array: A Disulfide Mapping and TAM-IDS Study
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Abstract
The
three core components of the ubiquitous bacterial chemosensory
array the transmembrane chemoreceptor, the histidine kinase
CheA, and the adaptor protein CheW 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 receptor–CheA 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. On–off
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