The crystal structure and mutational analysis of a novel RNA-binding domain found in the human Tap nuclear mRNA export factor.
The Tap protein mediates the sequence nonspecific nuclear export of cellular mRNAs as well as the sequence-specific export of retroviral mRNAs bearing the constitutive transport element (CTE). Previously, the structures of individual Tap subdomains, including ribonucleoprotein and leucine-rich repeat domains, have been described. Here, we report the crystal structure of a functional CTE RNA-binding domain of human Tap, including the N-terminal arm of the ribonucleoprotein domain and interdomain linking polypeptide. To identify residues that interact with the CTE, we have introduced 38 alanine substitutions for surface residues in the Tap CTE-binding domain and tested these mutants for their ability to support CTE-dependent nuclear RNA export and CTE binding. Four residues that cluster on a concave surface in the leucine-rich repeat domain were found to be critical for CTE binding and define a CTE-interacting surface on this domain. The second critical CTE-interacting surface on Tap is defined by three previously identified residues on the surface of the ribonucleoprotein domain. The structural and mutational data define a novel RNA-binding site on the Tap protein.
Ho
Dona N
DN
Waksman Institute and Department of Chemistry, Rutgers University, Piscataway, NJ 08854, USA.
Coburn
Glen A
GA
Kang
Yibin
Y
Cullen
Bryan R
BR
Georgiadis
Millie M
MM
D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics*; D029543:Nucleocytoplasmic Transport Proteins; D017434:Protein Structure, Tertiary; D011784:Quail; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics; D014162:Transfection
C122111:NXF1 protein, human; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012313:RNA