Abstract
The human PPIL1 (peptidyl prolyl isomerase-like protein 1) is a specific component of human 35 S U5 small nuclear ribonucleoprotein particle and 45 S activated spliceosome. It is recruited by SKIP, another essential component of 45 S activated spliceosome, into spliceosome just before the catalytic step 1. It stably associates with SKIP, which also exists in 35 S and activated spliceosome as a nuclear matrix protein. We report here the solution structure of PPIL1 determined by NMR spectroscopy. The structure of PPIL1 resembles other members of the cyclophilin family and exhibits PPIase activity. To investigate its interaction with SKIP in vitro, we identified the SKIP contact region by GST pulldown experiments and surface plasmon resonance. We provide direct evidence of PPIL1 stably associated with SKIP. The dissociation constant is 1.25 x 10(-7) M for the N-terminal peptide of SKIP-(59-129) with PPIL1. We also used chemical shift perturbation experiments to show the possible SKIP binding interface on PPIL1. These results illustrated that a novel cyclophilin-protein contact mode exists in the PPIL1-SKIP complex during activation of the spliceosome. The biological implication of this binding with spliceosome rearrangement during activation is discussed.
Highlights
The dissociation constant of 1.25 ϫ 10Ϫ7 M was obtained. These results reveal that PPIL1 can interact tightly with nSKIP-(59 – 129) in vitro
After superimposing PPIL1 to cyclophilin A, we found that side chains of these residues share a similar orientation and facilitate the formation of PPIL1-cyclosporin A complex
The results revealed that the nSKIP bound to SKIP tightly, which was consistent with the dissociation constant of ϳ10Ϫ7 M
Summary
Expression and Purification and Isotope Labeling of PPIL1—The hPPIL1 cDNA was obtained by PCR from human CD34ϩ hematopoietic stem/progenitor cell cDNA library cloned in-frame into the NdeI/ XhoI sites of pET-22b (ϩ) (Novagen). It was transformed into the Escherichia coli expression strain BL21 (DE3). Bacteria were grown at 37 °C in Luria Bertani medium containing 100 mg/liter of ampicillin. The pellet was resuspended in a buffer containing 50 mM phosphate, pH 7.5, 500 mM NaCl. Cells were disrupted by sonication, and debris was spun down at 15,000 rpm, 4 °C for 0.5 h in a Beckman centrifuge using a JA-17 rotor.
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