The aryl hydrocarbon receptor-interacting protein (AIP) has been predicted to consist of an N-terminal FKBP-type peptidyl-prolyl cis/trans isomerase (PPIase) domain and a C-terminal tetratricopeptide repeat (TPR) domain, as typically found in FK506-binding immunophilins. AIP, however, exhibited no inherent FK506 binding or PPIase activity. Alignment with the prototypic FKBP12 showed a high sequence homology but indicated inconsistencies with regard to the secondary structure prediction derived from chemical shift analysis of AIP(2-166). NMR-based structure determination of AIP(2-166) now revealed a typical FKBP fold with five antiparallel β-strands forming a half β-barrel wrapped around a central α-helix, thus permitting AIP to be also named FKBP37.7 according to FKBP nomenclature. This PPIase domain, however, features two structure elements that are unusual for FKBPs: (i) an N-terminal α-helix, which additionally stabilizes the domain, and (ii) a rather long insert, which connects the last two β-strands and covers the putative active site. Diminution of the latter insert did not generate PPIase activity or FK506 binding capability, indicating that the lack of catalytic activity in AIP is the result of structural differences within the PPIase domain. Compared to active FKBPs, a diverging conformation of the loop connecting β-strand C' and the central α-helix apparently is responsible for this inherent lack of catalytic activity in AIP. Moreover, Hsp90 was identified as potential physiological interaction partner of AIP, which revealed binding contacts not only at the TPR domain but uncommonly also at the PPIase domain.

Recently we have shown that the peptidyl-prolyl cis/trans isomerase parvulin 17 (Par17) interacts with tubulin in a GTP-dependent manner, thereby promoting the formation of microtubules. Microtubule assembly is regulated by Ca(2+)-loaded calmodulin (Ca(2+)/CaM) both in the intact cell and under in vitro conditions via direct interaction with microtubule-associated proteins. Here we provide the first evidence that Ca(2+)/CaM interacts also with Par17 in a physiologically relevant way, thus preventing Par17-promoted microtubule assembly. In contrast, parvulin 14 (Par14), which lacks only the first 25 N-terminal residues of the Par17 sequence, does not interact with Ca(2+)/CaM, indicating that this interaction is exclusive for Par17. Pulldown experiments and chemical shift perturbation analysis with (15)N-labeled Par17 furthermore confirmed that calmodulin (CaM) interacts in a Ca(2+)-dependent manner with the Par17 N terminus. The reverse experiment with (15)N-labeled Ca(2+)/CaM demonstrated that the N-terminal Par17 segment binds to both CaM lobes simultaneously, indicating that Ca(2+)/CaM undergoes a conformational change to form a binding channel between its two lobes, apparently similar to the structure of the CaM-smMLCK(796-815) complex. In vitro tubulin polymerization assays furthermore showed that Ca(2+)/CaM completely suppresses Par17-promoted microtubule assembly. The results imply that Ca(2+)/CaM binding to the N-terminal segment of Par17 causes steric hindrance of the Par17 active site, thus interfering with the Par17/tubulin interaction. This Ca(2+)/CaM-mediated control of Par17-assisted microtubule assembly may provide a mechanism that couples Ca(2+) signaling with microtubule function.

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Using quantitative phosphopeptide sequencing of unstimulated versus stimulated primary murine Foxp3+ regulatory and Foxp3– conventional T cells (Tregs and Tconv, respectively), we detected a novel and differentially regulated tyrosine phosphorylation site within the C1 domain of the guanine-nucleotide exchange factor CalDAG GEFI. We hypothesized that the Treg-specific and activation-dependent reduced phosphorylation at Y523 allows binding of CalDAG GEFI to diacylglycerol, thereby impacting the formation of a Treg-specific immunological synapse. However, diacylglycerol binding assays of phosphomutant C1 domains of CalDAG GEFI could not confirm this hypothesis. Moreover, CalDAG GEFI–/– mice displayed normal Treg numbers in thymus and secondary lymphoid organs, and CalDAG GEFI–/– Tregs showed unaltered in vitro suppressive capacity when compared to CalDAG GEFI+/+ Tregs. Interestingly, when tested in vivo, CalDAG GEFI–/– Tregs displayed a slightly reduced suppressive ability in the transfer colitis model when compared to CalDAG GEFI+/+ Tregs. Additionally, CRISPR-Cas9-generated CalDAG GEFI–/– Jurkat T cell clones showed reduced adhesion to ICAM-1 and fibronectin when compared to CalDAG GEFI-competent Jurkat T cells. Therefore, we speculate that deficiency in CalDAG GEFI impairs adherence of Tregs to antigen-presenting cells, thereby impeding formation of a fully functional immunological synapse, which finally results in a reduced suppressive potential.