Cyclophilin 1 (TvCyP1), a cyclophilin type peptidyl-prolyl isomerase present in the human parasite Trichomonas vaginalis, interacts with Myb1 and assists in its nuclear translocation. Myb1 regulates the expression of ap65-1 gene that encodes for a disease causing cytoadherence enzyme. Here, we determined the crystal structures of TvCyP1 and its complex with the minimum TvCyP1-binding sequence of Myb1 (Myb1104–111), where TvCyP1 formed a homodimer, unlike other single domain cyclophilins. In the complex structure, one Myb1104–111 peptide was bound to each TvCyP1 protomer, with G106-P107 and Y105 fitting well into the active site and auxiliary S2 pocket, respectively. NMR data further showed that TvCyP1 can catalyze the cis/trans isomerization of P107 in Myb1104–111. Interestingly, in the well-folded Myb1 protein (Myb135–141), the minimum binding sequence adopted a different conformation from that of unstructured Myb1104–111 peptide, that could make P107 binding to the active site of TvCyP1 difficult. However, NMR studies showed that similar to Myb1104–111 peptide, Myb135–141 also interacted with the active site of TvCyP1 and the dynamics of the Myb135–141 residues near P107 was reduced upon interaction. Together, the structure of TvCyP1 and detailed structural insights on TvCyP1-Myb1 interaction provided here could pave the way for newer drugs to treat drug-resistant strains.

Cyclophilins are peptidyl prolyl isomerases that play an important role in a wide variety of biological functions like protein folding and trafficking, intracellular and extracellular signaling pathways, nuclear translocation and in pre-mRNA splicing. Two cyclophilins have been identified in the parasitic organism Trichomonas vaginalis and were named as TvCyP1 and TvCyP2. The 2 enzymes have been found to interact with Myb transcription factors in the parasite which regulate the iron induced expression of ap65-1 gene leading to cytoadherence of the parasite to human vaginal epithelial cells to cause the disease trichomoniasis. TvCyP2 was found to interact specifically with Myb3 to regulate nuclear translocation of the transcription factor. It would be intriguing to identify the binding site of both proteins as it could pave way to newer targets for drug discovery. Here we report the H,C and N resonance assignments and secondary structure information of TvCyP2 that could help us investigate the interaction between Myb3 and TvCyP2 in detail using NMR.

Recent work has established that bacterial endotoxin (LPS) binds to the plasma protein LPS-binding protein (LBP) forming high affinity complexes (LPS-LBP), that LBP is an opsonin for LPS-bearing particles, and that LPS-LBP complexes are potent agonists for monocytic cells (MO). mAb to the MO plasma membrane protein, CD14, inhibit LBP-dependent binding of LPS to MO, and LPS-LBP-dependent stimulation of cytokine release from MO. These data suggest that CD14 functions as a membrane receptor for LPS but do not demonstrate a direct association of LPS with CD14. Calcitriol was used to induce a high level of CD14 expression in the human monocyte-like cell line THP-1, resulting in enhanced responses of these cells to LPS-LBP complexes manifested by enhanced binding of LPS and a decrease in the amount of LPS needed to induce IL-8 release. An Re595 LPS derivative containing a radioiodinated, photoreactive, phenyl azide (125I-ASD-LPS) was used in cross-linking experiments to identify membrane proteins in calcitriol-treated THP-1 cells that interact with LPS. 125I-ASD-LPS was added to calcitriol-induced THP-1 cells in the presence or absence of LBP, the mixture photolyzed, and the resultant radioiodinated proteins analyzed by SDS-PAGE and autoradiography. We observed strong cross-linking of 125I-ASD-LPS to a 55-kDa membrane protein when LBP was present, but failed to observe radiolabeling of any other proteins with apparent molecular masses distinct from CD14. The cross-linked product was identified as CD14 by immunoprecipitation with anti-human CD14 mAb. Studies with human CD14 expressing transfectants of the murine B cell line 70Z/3 also revealed LBP-dependent cross-linking of 125I-ASD-LPS to CD14. These data document binding of LPS to a specific membrane protein that serves as an LPS receptor.

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