Journal of Molecular Biology
Molecular Recognition of Human Eosinophil-derived Neurotoxin (RNase 2) by Placental Ribonuclease Inhibitor
Introduction
Accurate and highly selective recognition of proteins by other proteins drives most biological processes. However, there are numerous cases where an individual protein displays promiscuity in its ability to recognise multiple structurally related partners. Placental ribonuclease inhibitor (RI) is a remarkable example of such a promiscuous protein: it binds all of the different ∼14 kDa members of the mammalian pancreatic RNase superfamily with extraordinarily high avidity.1, 2, 3 The affinity of RI for its strongest-binding ligand, angiogenin (Ang; Ki=0.7 fM),4 is among the tightest on record for any protein-protein interaction; Ki values for other ligands also lie in the femtomolar range. These affinities are comparable to, or higher than, those measured for the complexes of TIMP with matrix metalloproteinases,5, 6 barnase with barstar,7 serpins with serine proteases,8 and colicins with their immunity proteins.9
Previous structural studies have shown that RI adopts a non-globular horseshoe shape10 and that the ligands RNase A and Ang occupy the central cavity of the horseshoe and contact one face of the inhibitor in the C-terminal region.11, 12 In both cases, the interface is large, encompassing 26–28 residues on the inhibitor and 24 on the ligand. Nonetheless, mutational analyses13, 14, 15 have revealed that in both complexes a single small region, containing the active site of the ligand and the 433–440 loop and C-terminal residue Ser460 of RI, contributes a substantial portion of the binding energy. In both cases, Asp435 of RI (human RI numbering) forms energetically important hydrogen bonds with the catalytic lysine of the ligand (Lys40 of Ang, Lys41 of RNase A). However, most of the other contacts in the two energetic “hot spots’16 do not correspond. Moreover, interactions within the hot spots exhibit opposite types of cooperativity in their functioning; thus, effects of multiple mutations in the Ang complex are largely superadditive, whereas in the RNase A complex they are subadditive.14, 15
A recent mutational study of the complex of human RI (hRI) with human eosinophil-derived neurotoxin (EDN; also known as RNase 2), suggests that this complex does not contain an analogous hot spot.17 Single-residue mutations of residues in the hRI C-terminal segment that had been shown to be functionally important in the Ang and RNase A complexes had much smaller effects, if any, on affinity for EDN. Individual replacements of RI residues in other regions, selected by examining a computational model of the hRI–EDN complex, did not implicate any other residues as playing key roles. Although multi-site mutagenesis revealed more sizable combined contributions by the C-terminal segment and a Trp-rich region of RI, most of the binding energy still could not be accounted for.
The three-dimensional structure of the hRI–EDN complex reported here provides a platform for a more complete understanding of the physical basis for high-affinity binding of EDN by RI. The structure reveals that EDN interacts with RI over a more extensive area than do RNase A and Ang and consequently forms a significantly larger number of intermolecular contacts. Mutational analysis based on this structure has now identified interactions unique to the hRI–EDN complex that make important contributions to the interface energetics. Our findings help explain how RI can bind such a diverse range of ligands with similarly high affinity.
Section snippets
Overall structure of the hRI–EDN complex
The hRI–EDN complex (Figure 1(a)) crystallises in the trigonal space group, P3121. The structure was determined at 2.0 Å resolution with two complexes (Complex 1 and Complex 2) in the asymmetric unit (see Table 1 for crystallographic statistics). The overall docking of EDN to hRI is similar to that of RNase A18 and Ang12 in their respective complexes with porcine RI (pRI) and hRI (Figure 1(b)). The characteristic modular architecture of hRI comprises 16 tandem alternating 28- and 29-residue
Structural basis for the recognition of different ligands by RI
RI was first characterized as an inhibitor of pancreatic RNase23 and was subsequently shown to bind extremely tightly to angiogenin,4, 24 EDN,25 and RNase 42, 26 as well. The range of Ki values measured for non-orthologous ligands, ∼1–200 fM, is remarkably small in view of the modest level of overall sequence identity between these proteins (∼25–40%)27 and the poor conservation of active site residues in particular (only the P1 catalytic site is universally maintained). In principle, RI might
Protein preparation, crystallisation and X-ray data collection
Natural hRI was obtained from Promega. Recombinant Met0 EDN was a kind gift from Dr R. J. Youle (NIH, Bethesda, MD). The hRI–EDN complex was prepared by a modification of the method used for the preparation of the pRI–RNase A complex.50 All buffers were degassed thoroughly before use. Briefly, hRI (0.9 mg) was mixed with EDN (0.4 mg) in 7.5 ml of 10 mM bis-Tris–HCl (pH 6.5), 20 mM DTT, and incubated on ice overnight. The mixture was injected onto a Mono-Q HR 5/5 anion-exchange column (Amersham
Acknowledgements
We thank the scientists at station PX 14.2, Synchrotron Radiation Source, Daresbury (UK) and the EMBL X11 beamline at the DORIS storage ring, DESY, Hamburg (Germany) for their support during X-ray data collection. This work was supported by the Wellcome Trust (UK) programme grant (067288) to K.R.A. and National Institutes of Health grant (CA-88738) to R.S. We would also like to acknowledge the support of the European Community, Research Infrastructure Action under the FP6 “Structuring the
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C-Mannosylation Enhances the Structural Stability of Human RNase 2
2020, iScienceCitation Excerpt :However, analysis of the beta factors indicates significant flexibility of the Trp7 side chain as well as the existence of several flexible loops (Figure 6). The loop with the highest beta factors (residues 87–96) is involved in binding of the placental ribonuclease inhibitor (Iyer et al., 2005). C-Mannosylation of Trp7 will most likely have an effect on the dynamics of the Trp7 side chain and the loop consisting of residues 115–123 (termed the “insertion loop” because these residues are not present in RNase 1) and/or the N terminus (residues 1–6) (Figure 6).
Charcot-Leyden crystal protein/galectin-10 interacts with cationic ribonucleases and is required for eosinophil granulogenesis
2020, Journal of Allergy and Clinical ImmunologyCrystal structure of human angiogenin with an engineered loop exhibits conformational flexibility at the functional regions of the molecule
2013, FEBS Open BioCitation Excerpt :This region is close to the active site of ANG and thus it was predicated that induction of similar conformational change would allow the C-terminal β-strand (Fig. 1A) to undergo small movement to accommodate the substrate/inhibitor without significant alteration in the catalytic property of the molecule. In this study we have substituted 84HGGSPWPP91 (a loop that interacts with RI) of ANG [39] with 86TTPSPQNISN95 of eosinophil-derived neurotoxin (EDN, also shown to interact with RI) [41] and constructed an ANG–EDN hybrid (AEH) protein molecule of molecular weight ∼14.5 kDa. EDN is also a member of RNase A superfamily (known as RNase 2), possesses weak ribonucleolytic activity, antiviral property [42–45] and neurotoxicity [46,47].
Degradation by stratum corneum proteases prevents endogenous RNase inhibitor from blocking antimicrobial activities of RNase 5 and RNase 7
2009, Journal of Investigative DermatologyCitation Excerpt :Both the candida–cidal activity of RNase 5 and the enterococcus–cidal activity of RNase 7 were effectively blocked by recombinant RI. Earlier reports have shown that RI suppresses the ribonuclease activity, which is low compared with other members of the RNase A family, and the angiogenic activity of RNase 5 (Shapiro et al., 1986; Shapiro and Vallee, 1987; Papageorgiou et al., 1997; Maeda et al., 2002; Iyer et al., 2005; Johnson et al., 2007). Two small chemical substances, DEPC and benzopurpurin B, which inhibit the catalytic activity of RNase 5, also suppressed its antimicrobial activity.
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Present address: K. Kumar, Biohelix Corporation, Beverly, MA 01915, USA.