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.

The Bcl-2 inhibitor FKBP38 is regulated by the Ca(2+)-sensor calmodulin (CaM). Here we show a hitherto unknown low-affinity cation-binding site in the FKBP domain of FKBP38, which may afford an additional level of regulation based on electrostatic interactions. Fluorescence titration experiments indicate that in particular the physiologically relevant Ca(2+) ion binds to this site. NMR-based chemical shift perturbation data locate this cation-interaction site within the β5-α1 loop (Leu90-Ile96) of the FKBP domain, which contains the acidic Asp92 and Asp94 side-chains. Binding constants were subsequently determined for K(+), Mg(2+), Ca(2+), and La(3+), indicating that the net charge and the radius of the ion influences the binding interaction. X-ray diffraction data furthermore show that the conformation of the β5-α1 loop is influenced by the presence of a positively charged guanidinium group belonging to a neighboring FKBP38 molecule in the crystal lattice. The position of the cation-binding site has been further elucidated based on pseudocontact shift data obtained by NMR via titration with Tb(3+). Elimination of the Ca(2+)-binding capacity by substitution of the respective aspartate residues in a D92N/D94N double-substituted variant reduces the Bcl-2 affinity of the FKBP38(35-153)/CaM complex to the same degree as the presence of Ca(2+) in the wild-type protein. Hence, this charge-sensitive site in the FKBP domain participates in the regulation of FKBP38 function by enabling electrostatic interactions with ligand proteins and/or salt ions such as Ca(2+).

The aryl-hydrocarbon receptor-interacting protein (AIP) interacts with several protein binding partners and has been associated with pituitary tumor development. Here, we report nearly complete (1)H, (13)C and (15)N chemical shift assignments for the N-terminal AIP(2-166) segment, which has been predicted to represent a FKBP-type PPIase domain. Sequence alignment with the prototypic FKBP12, however, reveals disagreements between the AIP chemical shift index consensus and the corresponding FKBP12 secondary structure elements.

The hsp70 chaperone DnaK from E. coli plays a major role in cellular stress response and is involved in assisted protein folding in vivo. By screening a combinatorial peptide library, we identified several DnaK‐specific peptide ligands with nanomolar affinities, which are able to inhibit the secondary amide peptide bond cis/trans isomerase (APIase) activity of DnaK, as well as DnaK/DnaJ/GrpE‐assisted refolding of firefly luciferase. Our designed DnaK inhibitors have the capability to penetrate E. coli cells and feature a high protease resistance. Once inside the cell, they physically target DnaK. NMR‐based 1H/15N‐HSQC experiments furthermore confirmed that the designed peptidic ligands all bind in an identical manner to the conventional peptide‐binding site of DnaK. The subsequent blocking of DnaK function apparently results in the observed antibacterial effects on E. coli cells, with minimum inhibitory concentrations in the range of 100 μM.

Background: FKBP38 regulates the prosurvival activity of Bcl-2 in apoptosis signaling. Results: The catalytic domain of FKBP38 interacts with helix 4 in Bcl-2. Conclusion: An electrostatic interaction modulates transient complex formation between FKBP38 and Bcl-2. Significance: The protein complex provides a molecular basis for FKBP38-mediated Bcl-2 regulation.