Fig. 4 compares the major sequences of FKBP12 and FKBP12.6 and their three-dimensional structuresFIGURE four Comparison of FKBP12 and FKBP12.six proteins to highlight the amino acid substitutions with the FKBP12E31Q/D32N/W59F mutant. (A) Amino acid sequence alignments of human FKBP12, human FKBP12.six, and FKBP12E31Q/D32N/W59F. The residues of FKBP12 and FKBP12.six that differ are highlighted in blue. The red arrows indicate the 3 amino acid residues that were substituted into the FKBP12E31Q/D32N/W59F mutant. (B) Overlaid ribbon diagrams of human FKBP12 and human FKBP12.six determined by their crystal structures (RCSB PDB accession codes 2DG3 and 1C9H, respectively) showing the three residues highlighted as most likely relevant amino acid substitutions among the two proteins. The dashes indicate these residues: Glu31 in FKBP12 is replaced by Gln31 from FKBP12.six (E31Q); Asp32 in FKBP12 is replaced by Asn32 from FKBP12.6 (D32N); Trp59 in FKBP12 is replaced by Phe59 from FKBP12.six (W59F). Mammalian FKBP12 and FKBP12.six are extremely conserved and Fig. S3 shows sequence alignment of various species demonstrating that the three residues that we chose to mutate were absolutely conserved in FKBP12 and have been diverse but, once more, definitely conserved in FKBP12.six.(FKBP12 purple; Protein Data Bank (PDB) 2DG3; FKBP12.six green; PDB 1C9H) and illustrates their high structural similarity. Indeed, the root mean-squared deviation amongst the superimposed backbone atoms is only ?0.44 A. Around the basis of their steric and electrostatic properties, we identified three residues that could have specific relevance. Glu31 and Asp32 are negatively charged residues of FKBP12, whereas the corresponding residues in FKBP12.six are neutral (Gln31 and Asn32). Trp59 in FKBP12 is situated within the hydrophobic binding pocket for rapamycin and has a bigger side chain (an indole) compared with Phe59 (a phenyl group) in FKBP12.5-Bromo-1H-pyrazolo[3,4-b]pyrazine supplier six.156311-83-0 Chemscene The locations of these three amino acid residues in FKBP12 and the corresponding residues in FKBP12.PMID:24293312 6 are highlighted.Biophysical Journal 106(4) 824?Venturi et al.We made a triple mutant of FKBP12 where these 3 highlighted amino acids were mutated towards the corresponding residues in FKBP12.six thus converting Glu31 to Gln, Asp32 to Asn, and Trp59 to Phe. The affinity of several mutants of FKBPs for RyR proteins have previously been investigated, (20,29) although the capacity to influence RyR single-channel function (efficacy) has not been studied. The FKBP12E31Q/ D32N/W59F triple mutant retains affinity for RyR channels (20,29) and consequently is often a molecule of selection for studying efficacy (mainly because we must use mutant molecules that bind to RyR channels to study efficacy). We then investigated the potential of FKBP12E31Q/D32N/W59F to influence the gating of RyR1 and RyR2 under identical experimental situations to those used together with the wild-type proteins. Representative examples of your effects of FKBP12E31Q/D32N/W59F on RyR2 gating are shown in Fig. 5. FKBP12E31Q/D32N/W59F developed no observable modifications in RyR2 gating even at nanomolar or micromolar concentrations. Clearly, the triple mutant protein, FKBP12E31Q/D32N/W59F, has lost the standard capacity of FKBP12 to activate RyR2 and behaves as an alternative like FKBP12.6; efficacy has been lost. When FKBP12E31Q/D32N/W59F was added to RyR1 channels, a rise in Po was observed (Fig. 6), pretty similar to that observed with FKBP12.6 (see Fig. 1). The representaAControlO CAO C O2 OControlPo=0.C0 0.20 Pocontrol FKBP12E31Q/D32N/W59FE31Q.
