The Cyclophilin A (CypA)/Apoptosis Inducing Factor (AIF) complex is implicated in the DNA degradation in response to various cellular stress conditions, such as oxidative stress, cerebral hypoxia-ischemia and traumatic brain injury. The pro-apoptotic form of AIF (AIF(Δ1-121)) mainly interacts with CypA through the amino acid region 370–394. The AIF(370-394) synthetic peptide inhibits complex formation in vitro by binding to CypA and exerts neuroprotection in a model of glutamate-mediated oxidative stress. Here, the binding site of AIF(Δ1-121) and AIF(370-394) on CypA has been mapped by NMR spectroscopy and biochemical studies, and a molecular model of the complex has been proposed. We show that AIF(370-394) interacts with CypA on the same surface recognized by AIF(Δ1-121) protein and that the region is very close to the CypA catalytic pocket. Such region partially overlaps with the binding site of cyclosporin A (CsA), the strongest catalytic inhibitor of CypA. Our data point toward distinct CypA structural determinants governing the inhibitor selectivity and the differential biological effects of AIF and CsA, and provide new structural insights for designing CypA/AIF selective inhibitors with therapeutic relevance in neurodegenerative diseases.

The complex formation between the proteins apoptosis-inducing factor (AIF) and cyclophilin A (CypA) following oxidative stress in neuronal cells has been suggested as a main target for reverting ischemia-stroke damage. Recently, a peptide encompassing AIF residues 370-394 has been developed to target the AIF-binding site on CypA, to prevent the association between the two proteins and suppress glutamate-induced cell death in neuronal cells. Using a combined approach based on NMR spectroscopy, synthesis and testing of all Ala-scan mutants of the peptide and molecular docking/molecular dynamics, we have generated a detailed model of the AIF (370-394)/CypA complex. The model suggests us that the central region of the peptide spanning residues V374-K384 mostly interacts with the protein and that for efficient complex inhibition and preservation of CypA activity, it is bent around amino acids F46-G75 of the protein. The model is consistent with experimental data also from previous works and supports the concept that the peptide does not interfere with other CypA activities unrelated to AIF activation; therefore, it may serve as an ideal template for generating future non-peptidic antagonists.

The cell surface receptor CD44 is a glycoprotein belonging to the hyaluronan-binding proteins, termed hyaladherins. CD44 is expressed in a wide variety of isoforms in many cells, and in particular is present on the surface of malignant cells where it is involved in the onset and progression of cancer. In a first attempt to identify novel CD44 binding agents, we first characterized via nuclear magnetic resonance (NMR) techniques, several agents that were reported to bind to hCD44. To our surprise, however, none of these putative CD44 binding agents including peptides, one of which is in phase 2 clinical trial (A6 peptide), and a recently reported fragment hit, were found to significantly interact with recombinant hCD44(21–178). Nonetheless, we further report that a fragment screening campaign, using solution NMR as detection method, identified a viable fragment hit that bound in a potentially functional pocket on the surface of CD44, opposite to the HA binding site. We hypothesize that this pocket could be indirectly associated with the cellular and in vivo activity of the A6 peptide hence providing a novel framework for a possible development of therapeutically viable CD44 antagonists.