Journal of Biomolecular NMR 29: 449–450, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands. 449 Letter to the Editor: Resonance assignment and secondary structure of the La motif Domenico Sanfelice a , Jeff Babon b , Geoff Kelly c , Stephen Curry d & Maria R. Conte a,∗ a Biophysics Laboratories, IBBS, University of Portsmouth, St. Michael’s Building, Portsmouth PO1 2DT, U.K.; b Department of Molecular Structure, Walter and Eliza Hall Institute, Parkville, Vic 3052, Australia; c Biomedical NMR Centre, National Institute for Medical Research, The Ridgeway, London NW7 1AA, U.K.; d Department of Biological Sciences, Blackett Laboratory, Imperial College of Science, Technology and Medicine, London SW7 2BW, U.K. Received 5 January 2004; Accepted 20 February 2004 Key words: La antigen, La motif, NMR assignment, RRM Biological context Processing of nascent RNA polymerase III (pol III) transcripts is a complex mechanism that involves the La protein, an abundant and evolutionarily con- served RNA-binding phosphoprotein first identified as an autoantigen in systemic lupus erythematosus and Sjogren’s syndrome patients (Maraia and Intine, 2001; Wolin and Cedervall, 2002). La acts by binding spe- cifically to the 3 ′ poly (U) ends of pol III precursors in an interaction mediated by the highly-conserved N- terminal domain (NTD) of the protein. La proteins of higher eukaryotes also possess a C-terminal domain responsible for RNA nuclear retention and recognition of the 5 ′ terminus of nascent RNA transcripts (Jacks et al., 2003; Maraia and Intine, 2001). The NTDs of La proteins contain two RNA bind- ing domains, both crucial for poly (U) RNA binding. These domains are predicted to be RNA recognition motifs (RRM) that bind with RNA via a four-strand β-sheet (Hall, 2002); however, whereas the domain spanning residues 105–194 has been shown to adopt an RRM-like structure ((Alfano et al., 2003) and un- published data), some authors contest that the first domain (residues 1–103) folds into a predominantly helical structure that has been termed a ‘La-motif’ (Wolin and Cedervall, 2002). Interestingly, the La motif is also conserved in other proteins otherwise un- related to bona fide La proteins, but its role remains uncertain (Wolin and Cedervall, 2002). In this note we present the essentially complete assignment for the ∗ To whom correspondence should be addressed. E-mail: sasi.conte@port.ac.uk La motif of human La (hLa) and the delineation of its secondary structure. Methods and experiments An N-terminal fragment of human La encompassing the residues 1–103 was subcloned by PCR into pET-30 expression vector using the LIC method- ology (Novagen). The hexahistidine-tagged recom- binant protein was overexpressed in BL21(DE3) pLysS E. coli cells grown minimal media containing 0.8 g·L −1 15 N-ammonium chloride and 2g·L −1 13 C glucose. Cell pellets were lysed in 20 mM Tris-HCl, 300 mM NaCl, 10 mM Imidazole, pH 8 and purified by affinity chromatography on a Ni-NTA resin (Qia- gen), following the manifacturer’s protocol. The pro- tein eluted was dialysed in 50 mM Tris-HCl, 0.2 mM EDTA, 1.5 mM MgCl 2 , 10% (v/v) glycerol pH 7.25 (buffer A), loaded on a 5 ml Hi-Trap Heparin column (Amersham-Pharmacia Biotech) and eluted with a lin- ear 0–2.0 M KCl gradient in buffer A. After dialysis in 50 mM Tris-HCl, 100 mM KCl, 5 mM Ca 2 Cl, pH 8 and cleavage with factor Xa (Novagen) to re- move the N-terminal hexahistidine tag, the samples were re-loaded onto the Ni-NTA column and the tag- free protein was collected in the flow-through. NMR samples were prepared dialysing the purified protein against a buffer containing 20 mM Tris-HCl, 100 mM KCl, 1 mM DTT, pH 7 which was then concentrated to 0.9 mM in 700 μl. NMR spectra were recorded at 293 K on Varian Inova spectrometers operating at 14.1 and 18.8 T. Sequence specific assignments for the backbone were accomplished using HNCA, HN(CO)CA, CBCANH, CBCA(CO)NH and HNCO experiments (Bax and Grzesiek, 1993). Side chain