Shape and oligomerization state of the cytoplasmic domain of the phototaxis transducer II from Natronobacterium pharaonis

Abstract

Phototaxis allows archaea to adjust flagellar motion in response to light. In the photophobic response of Natronobacterium pharaonis, light-activated sensory rhodopsin II causes conformational changes in the transducer II protein (pHtrII), initiating the twocomponent signaling system analogous to bacterial chemotaxis. pHtrII's cytoplasmic domain (pHtrII-cyt) is homologous to the cytoplasmic domains of eubacterial chemotaxis receptors. Chemotaxis receptors require dimerization for activity and are in vivoorganized in large clusters. In this study we investigated the oligomerization and aggregation states of pHtrII-cyt by using chemical cross-linking, analytical gel-filtration chromatography, and small-angle neutron scattering. We show that pHtrII-cyt is monomeric in dilute buffers, but forms dimers in 4 M KCl, the physiological salt concentration for halophilic archaea. At high ammonium sulfate concentration, the protein forms higher-order aggregates. The monomeric protein has a rod-like shape, 202 Å in length and 14.4 Å in diameter; upon dimerization the length increases to 248 Å and the diameter to 18.2 Å. These results suggest that under high salt concentration the shape and oligomerization state of pHtrII-cyt are comparable to those of chemotaxis receptors.archaebacteria ͉ dynamics ͉ halophilic ͉ small-angle neutron scattering

Expression of the halobacterial transducer protein HtrII from Natronomonas pharaonis in Escherichia coli

“…The effect of ionic strength is less expressed but still observable (5% increase at 1 M NaF). Remarkably, the data recently obtained for the cytoplasmic domain of NpHtrII [28] show great similarities to our data, especially concerning the dependency of the CD-spectra on the environmental salt concentration.…”

Flexibility of the Cytoplasmic Domain of the Phototaxis Transducer II from Natronomonas pharaonis

“…However, this shape is not consistent with CD data lacking evidence for helix altogether for the monomer, and indicating only 24% helix for the dimer in 4 M KCl [20]. Here, we provide evidence that despite “random coil” spectroscopic features, pHtrII-cyt can exist as a highly flexible, loosely packed but folded helical coiled-coil: (i) sequence-based structure predictions all strongly support a helical coiled-coil structure for pHtrII-cyt, and addition of TFE, a well-known helix inducing agent, is able to generate CD spectra that are characteristic for ~80% helix; (ii) both, uncharged electrolytes and salts induce negative ellipticity in CD spectra of pHtrII-cyt, but the thermal midpoint transitions occur at very low temperatures indicating thermal instability; (iii) the majority of signals in 1 H, 15 N-HSQC spectra are highly temperature-sensitive, with the largest number of visible peaks observed at low temperatures and best spectral dispersion in high salt at low temperatures.…”

“…These predictions are not consistent with the CD spectra recorded in the commonly used buffers, PBS, buffer A and buffer B, which were all indistinguishable and essentially lacked negative ellipticity at 222 nm characteristic for helix [20]. The values obtained from deconvolution of the spectra were ∼77% unstructured, ∼7% turn, ∼13% β -sheet, and ∼3% α -helix.…”

“…CD spectra were acquired using a Jasco 810 spectropolarimeter (Jasco Inc., Easton, Md, USA) as described previously [20]. Experimental data θ exp were recorded in a 0.2-cm quartz cell at pHtrII-cyt concentration of 0.1 mg/mL and then converted to the mean residue ellipticity according to [ θ ] MRW = θ exp  /(10* l * c * n ) where l is the path length of the cell in cm, c is the protein concentration in M, and n is the number of residues.…”

Biochemical characterization of the tandem HAMP domain from Natronomonas pharaonis as an intraprotein signal transducer

“…Because the sensory input of HtrII is probably only via intramembrane interactions with sensory rhodopsin II the lack of significant signal transducer properties of the N. pharaonis HAMP tandem or its single modules may have been due to specific structural or physicochemical requirements which are not met by the Tsr receptor. Another more remote possibility is that the HAMP domains of this halophile were unfolded in the more dilute buffers used here as conflicting data concerning this issue have been published . We therefore expressed several constructs without membrane domain, affinity purified the soluble constructs and examined protein folding by CD spectroscopy together with AC activity as loss of secondary structure is usually accompanied by loss of function.…”