14 research outputs found
Structural and chemical requirements for histidine phosphorylation by the chemotaxis kinase CheA
The CheA histidine kinase initiates the signal transduction pathway of bacterial chemotaxis by autophosphorylating a conserved histidine on its phosphotransferase domain (P1). Site-directed mutations of neighboring conserved P1 residues (Glu-67, Lys-48, and His-64) show that a hydrogen-bonding network controls the reactivity of the phospho-accepting His (His-45) in Thermotoga maritima CheA. In particular, the conservative mutation E67Q dramatically reduces phospho-transfer to P1 without significantly affecting the affinity of P1 for the CheA ATP-binding domain. High resolution crystallographic studies revealed that although all mutants disrupt the hydrogen-bonding network to varying degrees, none affect the conformation of His-45. N-15-NMR chemical shift studies instead showed that Glu-67 functions to stabilize the unfavored (NH)-H-delta 1 tautomer of His-45, thereby rendering the N-epsilon 2 imidazole unprotonated and well positioned for accepting the ATP phosphoryl group
Bacterial chemoreceptor arrays are hexagonally packed trimers of receptor dimers networked by rings of kinase and coupling proteins
Chemoreceptor arrays are supramolecular transmembrane machines of unknown structure that allow bacteria to sense their surroundings and respond by chemotaxis. We have combined X-ray crystallography of purified proteins with electron cryotomography of native arrays inside cells to reveal the arrangement of the component transmembrane receptors, histidine kinases (CheA) and CheW coupling proteins. Trimers of receptor dimers lie at the vertices of a hexagonal lattice in a âtwo-facing-twoâ configuration surrounding a ring of alternating CheA regulatory domains (P5) and CheW couplers. Whereas the CheA kinase domains (P4) project downward below the ring, the CheA dimerization domains (P3) link neighboring rings to form an extended, stable array. This highly interconnected protein architecture underlies the remarkable sensitivity and cooperative nature of transmembrane signaling in bacterial chemotaxis
Subunit Exchange by CheA Histidine Kinases from the Mesophile Escherichia coli and the Thermophile Thermotoga maritima
Dimerization of the chemotaxis histidine kinase CheA is required for intersubunit autophosphorylation [Swanson, R. V., Bourret, R. B., and Simon, M. I. (1993) Mol. Microbiol. 8, 435â441]. Here we show that CheA dimers exchange subunits by the rate-limiting dissociation of a central four-helix bundle association domain (P3), despite the high stability of P3 versus unfolding. P3 alone determines the stability and exchange properties of the CheA dimer. For CheA proteins from the mesophile Escherichia coli and the thermophile Thermotoga maritima, subunit dissociation activates at temperatures where the respective organisms live (37 and 80 °C). Under destabilizing conditions, P3 dimer dissociation is cooperative with unfolding. Chemical denaturation is reversible for both EP3 and TP3. Aggregation accompanies thermal unfolding for both proteins under most conditions, but thermal unfolding is reversible and two-state for EP3 at low protein concentrations. Residue differences within interhelical loops may account for the contrasted thermodynamic properties of structurally similar EP3 and TP3 (41% sequence identity). Under stabilizing conditions, greater correlation between activation energy for dimer dissociation and P3 stability suggests more unfolding in the dissociation of EP3 than TP3. Furthermore, destabilization of extended conformations by glycerol slows relative dissociation rates more for EP3 than for TP3. Nevertheless, at physiological temperatures, neither protein likely unfolds completely during subunit exchange. EP3 and TP3 will not exchange subunits with each other. The receptor coupling protein CheW reduces the subunit dissociation rate of the T. maritima CheA dimer by interacting with the regulatory domain P5
Subunit Exchange by CheA Histidine Kinases from the Mesophile Escherichia coli and the Thermophile Thermotoga maritima
Dimerization of the chemotaxis histidine kinase CheA is required for intersubunit autophosphorylation [Swanson, R. V., Bourret, R. B., and Simon, M. I. (1993) Mol. Microbiol. 8, 435â441]. Here we show that CheA dimers exchange subunits by the rate-limiting dissociation of a central four-helix bundle association domain (P3), despite the high stability of P3 versus unfolding. P3 alone determines the stability and exchange properties of the CheA dimer. For CheA proteins from the mesophile Escherichia coli and the thermophile Thermotoga maritima, subunit dissociation activates at temperatures where the respective organisms live (37 and 80 °C). Under destabilizing conditions, P3 dimer dissociation is cooperative with unfolding. Chemical denaturation is reversible for both EP3 and TP3. Aggregation accompanies thermal unfolding for both proteins under most conditions, but thermal unfolding is reversible and two-state for EP3 at low protein concentrations. Residue differences within interhelical loops may account for the contrasted thermodynamic properties of structurally similar EP3 and TP3 (41% sequence identity). Under stabilizing conditions, greater correlation between activation energy for dimer dissociation and P3 stability suggests more unfolding in the dissociation of EP3 than TP3. Furthermore, destabilization of extended conformations by glycerol slows relative dissociation rates more for EP3 than for TP3. Nevertheless, at physiological temperatures, neither protein likely unfolds completely during subunit exchange. EP3 and TP3 will not exchange subunits with each other. The receptor coupling protein CheW reduces the subunit dissociation rate of the T. maritima CheA dimer by interacting with the regulatory domain P5
Light-Induced Subunit Dissociation by a LightâOxygenâVoltage Domain Photoreceptor from <i>Rhodobacter sphaeroides</i>
Lightâoxygenâvoltage (LOV) domains bind
a flavin
chromophore to serve as blue light sensors in a wide range of eukaryotic
and prokaryotic proteins. LOV domains are associated with a variable
effector domain or a separate protein signaling partner to execute
a wide variety of functions that include regulation of kinases, generation
of anti-sigma factor antagonists, and regulation of circadian clocks.
Here we present the crystal structure, photocycle kinetics, association
properties, and spectroscopic features of a full-length LOV domain
protein from <i>Rhodobacter sphaeroides</i> (RsLOV). RsLOV
exhibits N- and C-terminal helical extensions that form an unusual
helical bundle at its dimer interface with some resemblance to the
helical transducer of sensory rhodopsin II. The blue light-induced
conformational changes of RsLOV revealed from a comparison of light-
and dark-state crystal structures support a shared signaling mechanism
of LOV domain proteins that originates with the light-induced formation
of a flavinâcysteinyl photoadduct. Adduct formation disrupts
hydrogen bonding in the active site and propagates structural changes
through the LOV domain core to the N- and C-terminal extensions. Single-residue
variants in the active site and dimer interface of RsLOV alter photoadduct
lifetimes and induce structural changes that perturb the oligomeric
state. Size exclusion chromatography, multiangle light scattering,
small-angle X-ray scattering, and cross-linking studies indicate that
RsLOV dimerizes in the dark but, upon light excitation, dissociates
into monomers. This light-induced switch in oligomeric state may prove
to be useful for engineering molecular associations in controlled
cellular settings
Nucleotide binding by the histidine kinase CheA
To probe the structural basis for protein histidine kinase (PHK) catalytic activity and the prospects for PHK-specific inhibitor design, we report the crystal structures for the nucleotide binding domain of Thermotoga maritima CheA with ADP and three ATP analogs (ADPNP, ADPCP and TNP-ATP) bound with either Mg2+ or Mn2+. The conformation of ADPNP bound to CheA and related ATPases differs from that reported in the ADPNP complex of PHK EnvZ. Interactions of the active site with the nucleotide bold gamma-phosphate and its associated Mg2+ ion are linked to conformational changes in an ATP-lid that could mediate recognition of the substrate domain. The inhibitor TNP-ATP binds CheA with its phosphates in a nonproductive conformation and its adenine and trinitrophenyl groups in two adjacent binding pockets. The trinitrophenyl interaction may be exploited for designing CheA-targeted drugs that would not interfere with host ATPases
The 3.2 Ă Resolution Structure of a Receptor:CheA:CheW Signaling Complex Defines Overlapping Binding Sites and Key Residue Interactions within Bacterial Chemosensory Arrays
Bacterial chemosensory arrays are
composed of extended networks
of chemoreceptors (also known as methyl-accepting chemotaxis proteins,
MCPs), the histidine kinase CheA, and the adaptor protein CheW. Models
of these arrays have been developed from cryoelectron microscopy,
crystal structures of binary and ternary complexes, NMR spectroscopy,
mutational, data and biochemical studies. A new 3.2 Ă
resolution
crystal structure of a <i>Thermotoga maritima</i> MCP protein
interaction region in complex with the CheA kinase-regulatory module
(P4âP5) and adaptor protein CheW provides sufficient detail
to define residue contacts at the interfaces formed among the three
proteins. As in a previous 4.5 Ă
resolution structure, CheA-P5
and CheW interact through conserved hydrophobic surfaces at the ends
of their ÎČ-barrels to form pseudo 6-fold symmetric rings in
which the two proteins alternate around the circumference. The interface
between P5 subdomain 1 and CheW subdomain 2 was anticipated from previous
studies, whereas the related interface between CheW subdomain 1 and
P5 subdomain 2 has only been observed in these ring assemblies. The
receptor forms an unexpected structure in that the helical hairpin
tip of each subunit has âunzippedâ into a continuous
α-helix; four such helices associate into a bundle, and the
tetramers bridge adjacent P5-CheW rings in the lattice through interactions
with both P5 and CheW. P5 and CheW each bind a receptor helix with
a groove of conserved hydrophobic residues between subdomains 1 and
2. P5 binds the receptor helix N-terminal to the tip region (lower
site), whereas CheW binds the same helix with inverted polarity near
the bundle end (upper site). Sequence comparisons among different
evolutionary classes of chemotaxis proteins show that the binding
partners undergo correlated changes at key residue positions that
involve the lower site. Such evolutionary analyses argue that both
CheW and P5 bind to the receptor tip at overlapping positions. Computational
genomics further reveal that two distinct CheW proteins in Thermotogae
utilize the analogous recognition motifs to couple different receptor
classes to the same CheA kinase. Important residues for function previously
identified by mutagenesis, chemical modification and biophysical approaches
also map to these same interfaces. Thus, although the native CheWâreceptor
interaction is not observed in the present crystal structure, the
bioinformatics and previous data predict key features of this interface.
The companion study of the P5-receptor interface in native arrays
(accompanying paper Piasta et al. (2013) <i>Biochemistry</i>, DOI: 10.1021/bi400385c) shows that, despite the non-native receptor
fold in the present crystal structure, the local helix-in-groove contacts
of the crystallographic P5-receptor interaction are present in native
arrays and are essential for receptor regulation of kinase activity