Fig. 7: CFAP45 mediates adenine nucleotide homeostasis via AMP binding. | Nature Communications

Fig. 7: CFAP45 mediates adenine nucleotide homeostasis via AMP binding.

From: CFAP45 deficiency causes situs abnormalities and asthenospermia by disrupting an axonemal adenine nucleotide homeostasis module

Fig. 7

a AMP-binding screen using AMP conjugated to agarose (blue circles) in four orientations (see also “Methods”). b CFAP45 immunoprecipitates from human sperm show affinity to AMP including 6AH-AMP-agarose following elution and IB with anti-CFAP45 antibody. c Coomasie-stained gel shows recombinant CFAP45- and AK8-enriched lysates recovered by TAP. d NTAP-CFAP45 shows affinity to AMP including 6AH-AMP-agarose by silver stain. e NTAP-AK8 shows affinity to αAH-AMP-agarose following elution and IB with monoclonal anti-FLAG antibody. f MT-sliding assay to reconstitute dynein ATPase activity. g 1 mM ATP or 1 mM ADP alone does not significantly reactivate sliding percentage of Cfap45−/− sperm compared to control (***p < 0.0001); however, 1 mM ADP significantly increases sliding percentage of Cfap45−/− sperm compared to 1 mM ATP (comparison of +/−, p = 0.3702, not significant; −/−, **p = 0.0042; data are means ± SEM; significance assessed by Fisher’s exact test); 1 mM AMP alone shows negligible (<1%) reactivation, similar to untreated control. h Combination of either 4 mM AMP (n = 113 sperm from 2 experiments, *p = 0.0432) or 4 mM ADP (n = 93 sperm over 2 experiments, p = 0.3781) with 1 mM ATP increases sliding percentage of Cfap45−/− sperm, compared to heterozygous control (data are the means ± SEM; significance supported by Fisher’s exact test); equimolar ratios of 1 mM AMP or 1 mM ADP with 1 mM ATP does not significantly reactivate Cfap45−/− sperm sliding percentage compared to control (data are means ± SEM; significance assessed by Fisher’s exact test; ***p < 0.0001). i Close-up view of AMP bound at the interface of CFAP45·AK8 complex. Carbon atoms in AMP, CFAP45, and AK8 are colored in green, yellow, and gray, respectively; nitrogen, oxygen, and phosphorus atoms are colored in blue, red, and orange, respectively. Distances shown by thin-dash lines are in Angstroms (Å). j Sequence alignments highlighting highly conserved CFAP45 residues Arg479 and Arg483- and AMP-binding site of AK8 (residues 354-357, black bar). Conserved residues highlighted in gray; arginine residues highlighted in orange. See also Supplementary Fig. 11 and “Methods”.

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