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Open Access Gold as soon as Publ. Version/Full Text is submitted to ZB.
Dimerization and protein binding specificity of the U2AF homology motif of the splicing factor Puf60.
J. Biol. Chem. 284, 630-639 (2009)
PUF60 is an essential splicing factor functionally related and homologous to U2AF(65). Its C-terminal domain belongs to the family of U2AF (U2 auxiliary factor) homology motifs (UHM), a subgroup of RNA recognition motifs that bind to tryptophan-containing linear peptide motifs (UHM ligand motifs, ULMs) in several nuclear proteins. Here, we show that the Puf60 UHM is mainly monomeric in physiological buffer, whereas its dimerization is induced upon the addition of SDS. The crystal structure of PUF60-UHM at 2.2 angstrom resolution, NMR data, and mutational analysis reveal that the dimer interface is mediated by electrostatic interactions involving a flexible loop. Using glutathione S-transferase pulldown experiments, isothermal titration calorimetry, and NMR titrations, we find that Puf60-UHM binds to ULM sequences in the splicing factors SF1, U2AF(65), and SF3b155. Compared with U2AF(65)-UHM, Puf60-UHM has distinct binding preferences to ULMs in the N terminus of SF3b155. Our data suggest that the functional cooperativity between U2AF(65) and Puf60 may involve simultaneous interactions of the two proteins with SF3b155.
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Publication type
Article: Journal article
Document type
Scientific Article
Keywords
n-15 nmr relaxation; staphylococcal alpha-toxin; backbone dynamics; structural basis; branch site; polypyrimidine tract; interaction network; rna-binding; factor sf1; recognition
ISSN (print) / ISBN
0021-9258
e-ISSN
1083-351X
Quellenangaben
Volume: 284,
Issue: 1,
Pages: 630-639
Publisher
American Society for Biochemistry and Molecular Biology
Reviewing status
Peer reviewed
Institute(s)
Institute of Structural Biology (STB)