Citrate-Binding Site in Proteorhodopsin Involves Two Lysines in the First Cytoplasmic Loop

Abstract

Citrate-induced aggregation of native proteorhodopsin (pR) from octylglucoside solutions, observed at citrate concentrations as low as ∼15 mM (pH 5.5-7), is fully reversible by removal of the citrate and raising of the pH in the presence of detergent. The aggregation is specific for several tricarboxylic acid (TCA) cycle intermediates, including citrate, isocitrate, and cis-aconitate. The aggregation is not merely salting-out, since it is inhibited by phosphate. The citrate binding site on pR includes lysines 57 and 59, two of the four cationic residues in the first intracellular loop. That is, citrate no longer causes precipitation of pR when these lysines are mutated. The loss of citrate sensitivity is nearly complete for the quadruple mutant R51Q/R53Q/K57Q/K59Q, even with 1 M citrate. The double mutant K57Q/K59Q behaves nearly the same as the quadruple mutant. With the single-site R51Q mutant, on the other hand, there is only a ∼2-fold increase in the citrate concentration required to induce pR aggregation. Thus, the 2 arginines in the 1st intracellular loop play only a minor role in the citrate binding site Lysines 57 and 59 are conserved among pR sequences from genetically diverse /gamma/-proteobacteria. This conservation likely reflects a physiological function for the citrate binding site of pR, i.e. a structure-function relationship. Aggregation of pR occurs at citrate concentrations only a bit above the endogenous concentration, ∼10 mM, that has been measured inside thriving bacterial cells. This suggests that pR activity may be allosterically regulated by intracellular citrate. This would be consistent with recent observations in other labs, that bacteria expressing pR carry out light energy transduction only under starvation conditions that would be expected to decrease intracellular tricarboxylic acid concentrations.