Differential radial capillary action of ligand assay for high-throughput detection of protein-metabolite interactions

Abstract

Interactions of proteins with low-molecular-weight ligands, such as metabolites, cofactors, and allosteric regulators, are important determinants of metabolism, gene regulation, and cellular homeostasis. Pharmaceuticals often target these interactions to interfere with regulatory pathways. We have developed a rapid, precise, and high-throughput method for quantitatively measuring protein-ligand interactions without the need to purify the protein when performed in cells with low background activity. This method, differential radial capillary action of ligand assay (DRaCALA), is based on the ability of dry nitrocellulose to separate the free ligand from bound protein-ligand complexes. Nitrocellulose sequesters proteins and bound ligand at the site of application, whereas free ligand is mobilized by bulk movement of the solvent through capillary action. We show here that DRaCALA allows detection of specific interactions between three nucleotides and their cognate binding proteins. DRaCALA allows quantitative measurement of the dissociation constant and the dissociation rate. Furthermore, DRaCALA can detect the expression of a cyclic-di-GMP (cdiGMP)-binding protein in whole-cell lysates of Escherichia coli, demonstrating the power of the method to bypass the prerequisite for protein purification. We have used DRaCALA to investigate cdiGMP signaling in 54 bacterial species from 37 genera and 7 eukaryotic species. These studies revealed the presence of potential cdiGMP-binding proteins in 21 species of bacteria, including 4 unsequenced species. The ease of obtaining metabolite-protein interaction data using the DRaCALA assay will facilitate rapid identification of protein-metabolite and protein-pharmaceutical interactions in a systematic and comprehensive approach.

Fig. 1.

Principle of DRaCALA. (A) Schematic representation of DRaCALA assay on application of protein-ligand mixture onto nitrocellulose and capillary action. Protein (P), ligand (L), and protein–ligand complex (P•L) distribution during the assay is shown. (B) Equations are used to analyze DRaCALA data for F_B for purified proteins. An explanation of the apparent edge effect at the capillary migration front is provided in Fig. S3.

Fig. 2.

Detection of specific protein-ligand interactions by DRaCALA. (A) DRaCALA images of interactions between purified proteins (20 μM) incubated with 500 nM ¹⁴C-cAMP, 4 nM [³²P]ATP, or 4 nM ³²P-cdiGMP. Protein-ligand mixtures were spotted on nitrocellulose and allowed to dry before imaging using a Fuji FLA7100 PhosphorImager. Cognate protein-nucleotide combinations are indicated by arrowheads. MBP was used as a negative control. (B) DRaCALA images of competition assays assessing the ability of 1 mM indicated cold nucleotides to compete with binding interactions between 4 nM ³²P-cdiGMP and 2.5 μM HisMBP-Alg44_PilZ. (C) Graph of F_B for each sample in Fig. 1B, with averages indicated by a horizontal bar. NC, no competitor. P values were determined by a Student t test for significant differences compared with the NC control for three independent experiments. The I_total of each DRaCALA spot in Fig. 2 A and B is provided in Tables S1 and S2, respectively.

Fig. 3.

Determination of K_d and k_off by DRaCALA. (A) DRaCALA images were used for K_d determination for the interaction of Alg44_PilZ and cdiGMP. His-MBP-Alg44_PilZ was varied from 100 μM to 6 nM, and the ³²P-cdiGMP was held constant at 4 nM. Representative images of six sets of DRaCALA experiments are shown for 40 nM to 25 μM Alg44 protein. (B) F_B from data in A were plotted as a function of [MBP-Alg44_PilZ], and the best-fit line was determined by nonlinear regression using the indicated equation. A no-protein control was also plotted. The fitting program varied both K_d and B_max to obtain the best fit, indicated by the solid line. (C) k_off was determined by spotting protein-ligand mixtures onto nitrocellulose at various times after the addition of 1 mM cold cdiGMP to a mixture of 4 nM ³²P-cdiGMP and HisMBP-Alg44_PilZ. (D) Time course of decrease of F_B from analysis of the data in C was fitted to a single exponential decay, indicating k_off. The I_total of each DRaCALA spot in Fig. 3 A and C is provided in Tables S3 and S4, respectively.

Fig. 4.

Detection of specific protein-ligand interaction in whole-cell lysates by DRaCALA. (A) Images of Alg44_PilZ interaction with 4 nM ³²P-cdiGMP and either 1 mM cold cdiGMP or GTP with purified proteins or when expressed in E. coli BL21(DE3). C, cdiGMP; G, GTP. (B) Graph of ³²P-cdiGMP binding by whole-cell lysate samples (○) and purified proteins (▼) in Fig. 4A, with the average indicated by a horizontal bar. P values were determined by a Student t test for significant differences compared with the no-competitor control for three independent experiments. The I_total of each DRaCALA spot in Fig. 4B is provided in Tables S5 and S6. (C) Graph of ³²P-cdiGMP binding by purified MBP-Alg44_PilZ, purified MBP-Alg44_PilZ added to BL21 whole-cell lysates, and whole-cell lysates of BL21(DE3) overexpressing MBP-Alg44_PilZ. Protein concentrations were determined by separation on SDS/PAGE and staining with Coomassie blue (Fig. S4).

Fig. 5.

Analysis of cdiGMP-binding proteins in various organisms. B_Sp values of whole-cell lysates are shown as a heat map using the range indicated in the legend. (A) Equations used to analyze DRaCALA data for B_Sp for whole-cell lysates or tissue extracts. (B) Plate 1 is the analysis of cdiGMP binding by lysates from P. aeruginosa isolates. Specific strains discussed in the text are indicated by arrows. Sources of all strains in plates 1 and 2, as well as the raw data for each lysate, are shown in Table S7. (C) Plate 3 is the analysis of cdiGMP binding by lysates from various organisms. Plate numbers, column numbers, and row letters correspond to the strains and organisms listed in Tables S7 and S8.