Sequence-specific backbone (1)H, (13)C, and (15)N resonance assignments of human ribonuclease 4

Abstract

Human ribonuclease 4 (RNase 4) is the most evolutionarily conserved member of the 8 canonical human pancreatic-like RNases, showing more than 90% identity with bovine and porcine homologues. The enzyme displays ribonucleolytic activity with a strong preference for uracil-containing RNA substrates, a feature only shared with human eosinophil derived-neurotoxin (EDN, or RNase 2) and eosinophil cationic protein (ECP, or RNase 3). It is also the shortest member of the human family, with a significantly truncated C-terminal tail. Its unique active-site pocket and high degree of conservation among vertebrates suggest that the enzyme plays a crucial biological function. Here, we report on the (1)H, (13)C and (15)N backbone resonance assignments of RNase 4, providing means to characterize its molecular function at the atomic level by NMR.

Fig. 1

Sequence alignment of RNase 4 members found among vertebrates. Alignment was performed with T-Coffee (Notredame et al., 2000) using the following orthologues: Homo sapiens (human), Pan troglodytes (chimpanzee), Bos taurus (cattle), Sus scrofa (wild boar), Rattus norvegicus (brown rat) and Mus musculus (mouse). Residues showing >90% identity (similarity) are highlighed in black (grey). Alignment was processed with BoxShade 3.21.

Fig. 2

Schematic representation of human RNase 4. Three-dimensional view of RNase 4 (PDB entry 1RNF) highlighting the approximate active-site position of 3′-UMP (red), resulting from a structural overlay with RNase A-3′-UMP (PDB entry 1O0N). Catalytic residues His12, Lys40, His116 and Lys65 are shown as blue sticks. PyMOL was used to build all structures (Sayle and Milner-White, 1995).

Fig. 3

Two-dimensional ¹H–¹⁵N HSQC spectrum of human RNase 4. All assigned cross peaks have been labeled with the single letter amino acid code along with the sequence specific residue number.

Fig. 4

Evolutionary conservation of amino acid positions among vertebrate RNase 4 members. Residue positions are colored by their conservation grades using the color-coding bar, with turquoise-through-maroon indicating variable-through-conserved residues. Color ramping is identified on (A) the structure and (B) the amino acid sequence of RNase 4 (PDB entry 1RNF). The figure was generated using a PyMOL script generated by the ConSurf Server.