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In halophilic archaea the photophobic response is mediated by the membrane-embedded 2:2 photoreceptor/-transducer complex SRII/HtrII, the latter being homologous to the bacterial chemoreceptors. Both systems bias the rotation direction of the flagellar motor via a two-component system coupled to an extended cytoplasmic signaling domain formed by a four helical antiparallel coiled-coil structure. For signal propagation by the HAMP domains connecting the transmembrane and cytoplasmic domains, it was suggested that a two-state thermodynamic equilibrium found for the first HAMP domain in NpSRII/NpHtrII is shifted upon activation, yet signal propagation along the coiled-coil transducer remains largely elusive, including the activation mechanism of the coupled kinase CheA. We investigated the dynamic and structural properties of the cytoplasmic tip domain of NpHtrII in terms of signal transduction and putative oligomerization using site-directed spin labeling electron paramagnetic resonance spectroscopy. We show that the cytoplasmic tip domain of NpHtrII is engaged in a two-state equilibrium between a dynamic and a compact conformation like what was found for the first HAMP domain, thus strengthening the assumption that dynamics are the language of signal transfer. Interspin distance measurements in membranes and on isolated 2:2 photoreceptor/transducer complexes in nanolipoprotein particles provide evidence that archaeal photoreceptor/-transducer complexes analogous to chemoreceptors form trimers-of-dimers or higher-order assemblies even in the absence of the cytoplasmic components CheA and CheW, underlining conservation of the overall mechanistic principles underlying archaeal phototaxis and bacterial chemotaxis systems. Furthermore, our results revealed a significant influence of the NpHtrII signaling domain on the NpSRII photocycle kinetics, providing evidence for a conformational coupling of SRII and HtrII in these complexes.

Recent kinetic studies revealed distinct modes of inhibition of mitochondrial Arabidopsis thaliana succinic semialdehyde dehydrogenase (At-SSADH1) by AMP and ATP. Inhibition of SSADH by ATP may represent an important mechanism of feedback regulation of the GABA shunt by the respiratory chain. Here we used two approaches to investigate the interaction of ATP with At-SSADH1. Cofactor displacement studies based on the reduced fluorescence intensity of free NADH versus that of enzyme-bound NADH revealed that both AMP and ATP decreased NADH-At-SSADH1 complex formation. The competitive inhibitor AMP displaced all bound NADH, while ATP, a noncompetitive inhibitor, could not, even in great excess, release all NADH from its binding site. To assess the effect of ATP on NAD-At-SSADH, we employed surface plasmon resonance to monitor nucleotide binding to immobilized At-SSADH1. For this, we used a Strep-tag II modified derivative of At-SSADH1 (designated ST-At-SSADH1). The tagged enzyme was tightly and reversibly captured by StrepTactin, which was covalently immobilized on a CM5 chip. The binding constants for NAD(+) and ATP were determined from titration curves and were in good agreement with the constants obtained from enzyme kinetics. Surface plasmon resonance measurements confirmed that ATP binds to a site different from the binding site for NAD(+). GTP competed with ATP. However, only ATP increased the dissociation constant of NAD(+) from SSADH. This explains the reduced affinity of NAD(+)/NADH to At-SSADH1 in the presence of ATP, as revealed by enzymatic kinetics, and supports our model of feedback regulation of SSADH and the GABA shunt by ATP.