The first crystal structure of human RNase 6 reveals a novel substrate-binding and cleavage site arrangement

Abstract

Human RNase 6 is a cationic secreted protein that belongs to the RNase A superfamily. Its expression is induced in neutrophils and monocytes upon bacterial infection, suggesting a role in host defence. We present here the crystal structure of RNase 6 obtained at 1.72 Å (1 Å=0.1 nm) resolution, which is the first report for the protein 3D structure and thereby setting the basis for functional studies. The structure shows an overall kidney-shaped globular fold shared with the other known family members. Three sulfate anions bound to RNase 6 were found, interacting with residues at the main active site (His(15), His(122) and Gln(14)) and cationic surface-exposed residues (His(36), His(39), Arg(66) and His(67)). Kinetic characterization, together with prediction of protein-nucleotide complexes by molecular dynamics, was applied to analyse the RNase 6 substrate nitrogenous base and phosphate selectivity. Our results reveal that, although RNase 6 is a moderate catalyst in comparison with the pancreatic RNase type, its structure includes lineage-specific features that facilitate its activity towards polymeric nucleotide substrates. In particular, enzyme interactions at the substrate 5' end can provide an endonuclease-type cleavage pattern. Interestingly, the RNase 6 crystal structure revealed a novel secondary active site conformed by the His(36)-His(39) dyad that facilitates the polynucleotide substrate catalysis.

Keywords: RNase A superfamily; RNase k6; kinetic characterization; molecular dynamics; protein crystallography; sulfate anion.

Figure 1. Primary structure of human RNases and 3D structure of RNase 6 coloured by residue conservation score

(A) Structure-based sequence of the eight canonical human RNases together with RNase A. The active sites are highlighted in yellow. The four disulfide bonds are labelled with green numbers. Tested mutations on RNase 6 are indicated with red arrows. The alignment was performed using ClustalW, and drawn using ESPript (http://espript.ibcp.fr/ESPript/). Labels are as follows: red box, white character for strict identity; red character for similarity in a group and character with a blue frame for similarity across groups. (B) RNase 6 3D structure surface representation using the CONSURF web server (http://consurf.tau.ac.il/) featuring the relationships among the evolutionary conservation of amino acid positions within the RNase A family. The 3D structure shows residues coloured by their conservation score using the colour-coding bar at the bottom. Sulfate anions (S1–S4) and the glycerol (GOL) molecule found in the crystal structure are depicted. Conserved residues belonging to the RNase catalytic site and interacting with bound sulfate anions are labelled.

Figure 2. Detail of sulfate binding interactions in the RNase 6 crystal structure

Atoms involved in the protein–anion interactions are listed in Supplementary Table S3. The sulfate involved in the crystal packing (S3) is not shown. The structure was drawn with PyMol 1.7.2 (DeLano Scientific).

Figure 3. Illustrative scheme of RNase 6 main and putative secondary active sites

Illustrative scheme of the RNase 6 main active site (A) and the putative secondary site (B). CpA atom distances are labelled together with the hisitidine ND1 to NE2 respective distances. The figure was created with PyMol (DeLano Scientific).

Figure 4. Predicted RNase 6 and RNase A structures in complex with dinucleotides

Predicted structure of RNase 6 and RNase A in complex with CpA, UpA and UpG dinucleotides after MD simulations, as detailed in the Materials and methods section. Nucleotides are coloured green. RNases interacting residues are coloured magenta. Hydrogen bonds are coloured yellow. Structures were drawn with UCSF Chimera 1.10 [77].

Figure 5. Stereo view of predicted RNase 6 heptanucleotide complex

RNase 6 in complex with CCCAUAA heptanucleotide after a MD simulation, as described in the Materials and methods section. The heptanucleotide is coloured green. Interacting residues of RNase 6 are coloured turquoise. Protein-interacting residues and ligand atoms are coloured according to their element. Hydrogen bonds are coloured yellow. Overlapped sulfate ions of the original coordinates of the crystal are coloured magenta. The structures were drawn with UCSF Chimera 1.10 [77].

Figure 6. Analysis of polynucleotide cleavage pattern by RNase 6 and RNase A

Poly(C) cleavage pattern obtained by RNase 6 (A) compared with RNase A [27] (B). Chromatography profiles of poly(C) digestion products are shown at selected incubation times corresponding to representative steps of the catalysis process. See the Materials and methods section for substrate digestion conditions.

Figure 7. Analysis of polynucleotide cleavage pattern by RNase 6 mutants

Poly(C) cleavage pattern by RNase 6-H15A (A) and RNase 6-H36R (B) mutants. Chromatography profiles of poly(C) digestion products are shown at selected incubation times corresponding to representative steps of the catalysis process.