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J Biomol NMR (2015) 61:8387 DOI 10.1007/s10858-014-9878-3
NMR STRUCTURE NOTE
NMR structure determination of the protein NP_344798.1 as the rst representative of Pfam PF06042
Biswaranjan Mohanty Pedro Serrano
Michael Geralt Kurt Wthrich
Received: 26 September 2014 / Accepted: 17 November 2014 / Published online: 28 November 2014 Springer Science+Business Media Dordrecht 2014
Biological context
The protein NP_344798.1 from Streptococcus pneumoniae (TIGR4) belongs to the Pfam protein family PF06042, which currently contains 786 sequences from 739 species (http://pfam.xfam.org/family/PF06042
Web End =http:// http://pfam.xfam.org/family/PF06042
Web End =pfam.xfam.org/family/PF06042 ). Based on sequence analyses, NP_344798.1 has been identied as a member of the nucleotidyltransfererase (NTase)-fold superfamily and annotated as a protein domain of unknown function, DUF925 (Kuchta et al. 2009). The bioinformatics data further suggest that all PF06042 members contain a single domain and are active NTases, as they contain the characteristic functional catalytic residues (Kuchta et al. 2009). Nonetheless, due to scarcity of experimental data, no specic biological role of these enzymes could as yet be ascertained. The NMR core of the Joint Center for Structural Genomics (JCSG) targeted NP_344798.1 to obtain a rst structure for the PF06042 family, and the J-UNIO protocol for automated NMR
structure determination (Serrano et al. 2012) was applied to this 191-residue protein. The structure now presents a foundation for obtaining new insights into the function of proteins in this family by structure comparison with other related structures in the PDB, experimental studies of substrate binding and specicity, and homology modeling of other proteins in PF06042.
Methods
Protein preparation
The protein NP_344798.1 was expressed in E. coli, using the pSpeedET-NP_344798.1 plasmid produced by the JCSG crystallomics core, and was puried following our standard protocol (Serrano et al. 2012). Micro-scale exploratory experiments were pursued with the [u-15N]-
protein (Pedrini et al. 2013; Serrano et al. 2012). For the NMR structure determination, a 1.2 mM solution of the [u-13C,15N]-labeled protein was prepared, with 20 mM sodium phosphate at pH 6.0, 50 mM sodium chloride and4.5 mM NaN3 in 5 % (v/v) D2O/95 % H2O. To remove oxygen prior to data collection, the sample was treated with Argon gas in the NMR tube.
NMR spectroscopy
2D [15N,1H]-HSQC spectra and APSY-NMR datasets (Hiller et al. 2005, 2008) were recorded on a Bruker Avance 600 MHz spectrometer equipped with...