N-Terminal Segment of TvCyP2 Cyclophilin from Trichomonas vaginalis Is Involved in Self-Association, Membrane Interaction, and Subcellular Localization

Abstract

In Trichomonas vaginalis (T. vaginalis), cyclophilins play a vital role in dislodging Myb proteins from the membrane compartment and leading them to nuclear translocation. We previously reported that TvCyP1 cyclophilin from T. vaginalis forms a dimer and plays an essential role in moving the Myb1 transcription factor toward the nucleus. In comparison, TvCyP2 containing an extended segment at the N-terminus (N-terminal segment) formed a monomer and showed a different role in regulating protein trafficking. Four X-ray structures of TvCyP2 were determined under various conditions, all showing the N-terminal segment interacting with the active site of a neighboring TvCyP2, an unusual interaction. NMR study revealed that this particular interaction exists in solution as well and also the N-terminal segment seems to interact with the membrane. In vivo study of TvCyP2 and TvCyP2-∆N (TvCyP2 without the N-terminal segment) indicated that both proteins have different subcellular localization. Together, the structural and functional characteristics at the N-terminal segment offer valuable information for insights into the mechanism of how TvCyP2 regulates protein trafficking, which may be applied in drug development to prevent pathogenesis and disease progression in T. vaginalis infection.

Comprehensive Workflow for Mapping Disulfide Linkages Including Free Thiols and Error Checking by On-Line UV-Induced Precolumn Reduction and Spiked Control

“…In comparison, r Tv CyP2 was prepared and analyzed using method 3. UV-LC–MS 2 data indicated that r Tv CyP2 also contains a high thiol percentage (Table S4), consistent with the reported structure . However, as shown in Figure D, one disulfide cluster, Cy2T1–Cy2T3, was detected with insignificant control–target mispairings.…”

“…UV-LC−MS 2 data indicated that rTvCyP2 also contains a high thiol percentage (Table S4), consistent with the reported structure. 35 However, as shown in Figure 4D, one disulfide cluster, Cy2T1−Cy2T3, was detected with insignificant control−target mispairings. More importantly, the identified C53−C181 disulfide contained in Cy2T1−Cy2T3 of rTvCyP2 (Figure S13) is conserved in rTvCyP1 which exists as C41−C169 in disulfide pattern 1 (C41−C169/C164−C153 and C135 free) of rTvCyP1.…”

“…34 In comparison, rTvCyP2 which shares 69% sequence identity with rTvCyP1 (Figure S9) was also prepared and analyzed. rTvCyP2 contains three cysteine residues which are also far away in forming disulfide linkages, 35 two (C53 and C181) of which are conserved in rTvCyP1 (C41 and C169).…”

Structural Basis for Cyclosporin Isoform-Specific Inhibition of Cyclophilins from Toxoplasma gondii

“…In particular, the sequence comparison of TgCyp23 with human CypA suggested that, among the 13 residues characterizing the CsA-binding pocket in the human CypA–CsA complex (Arg55, Phe60, Met61, Gln63, Gly72, Ala101, Asn102, Ala103, Gln111, Phe113, Trp121, Leu122, His126), 12 are identical in TgCyp23, including a single tryptophan that is likely to be critical for CsA binding. However, TgCyp23 possesses a 40-residue N-terminal extension relative to human CypA (Figure ) that might be involved in the membrane interaction and subcellular localization in agreement to other parasitic systems . On the other hand, TgCyp18.4 contains some striking amino acid substitutions in the active site (e.g., Met61, Trp121, and His126 of CypA are replaced by Ala50, His111, and Tyr116, respectively, in TgCyp18.4), which could, in principle, influence the catalytic properties of the enzyme and its affinity for CsA, making it an attractive candidate for the design of new CsA derivative compounds (Figure ).…”

“…However, TgCyp23 possesses a 40-residue N-terminal extension relative to human CypA ( Figure 1 ) that might be involved in the membrane interaction and subcellular localization in agreement to other parasitic systems. 32 On the other hand, TgCyp18.4 contains some striking amino acid substitutions in the active site (e.g., Met61, Trp121, and His126 of CypA are replaced by Ala50, His111, and Tyr116, respectively, in TgCyp18.4), which could, in principle, influence the catalytic properties of the enzyme and its affinity for CsA, making it an attractive candidate for the design of new CsA derivative compounds ( Figure 1 ).…”