Human regulatory protein Ki-1/57 has characteristics of an intrinsically unstructured protein.
The human protein Ki-1/57 was first identified through the cross reactivity of the anti-CD30 monoclonal antibody Ki-1, in Hodgkin lymphoma cells. The expression of Ki-1/57 in diverse cancer cells and its phosphorylation in peripheral blood leukocytes after mitogenic activation suggested its possible role in cell signaling. Ki-1/57 interacts with several other regulatory proteins involved in cellular signaling, transcriptional regulation and RNA metabolism, suggesting it may have pleiotropic functions. In a previous spectroscopic analysis, we observed a low content of secondary structure for Ki-1/57 constructs. Here, Circular dichroism experiments, in vitro RNA binding analysis, and limited proteolysis assays of recombinant Ki-1/57(122-413) and proteolysis assays of endogenous full length protein from human HEK293 cells suggested that Ki-1/57 has characteristics of an intrinsically unstructured protein. Small-angle X-ray scattering (SAXS) experiments were performed with the C-terminal fragment Ki-1/57(122-413). These results indicated an elongated shape and a partially unstructured conformation of the molecule in solution, confirming the characteristics of an intrinsically unstructured protein. Experimental curves together with ab initio modeling approaches revealed an extended and flexible molecule in solution. An elongated shape was also observed by analytical gel filtration. Furthermore, sedimentation velocity analysis suggested that Ki-1/57 is a highly asymmetric protein. These findings may explain the functional plasticity of Ki-1/57, as suggested by the wide array of proteins with which it is capable of interacting in yeast two-hybrid interaction assays.
Bressan
Gustavo C
GC
National Synchrotron Light Laboratory - LNLS, 13083-970, Campinas, SP, Brazil.
Silva
Júlio C
JC
Borges
Júlio C
JC
Dos Passos
Dario O
DO
Ramos
Carlos H I
CH
Torriani
Iris L
IL
Kobarg
Jörg
J
D000595:Amino Acid Sequence; D002460:Cell Line; D019286:Endopeptidase K / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D018006:Myogenic Regulatory Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D053838:Scattering, Small Angle; D015398:Signal Transduction
C500946:HABP4 protein, human; D018006:Myogenic Regulatory Factors; D019286:Endopeptidase K