TY - JOUR
T1 - Exploring the RNase A scaffold to combine catalytic and antimicrobial activities. Structural characterization of RNase 3/1 chimeras
AU - Fernández-Millán, Pablo
AU - Vázquez-Monteagudo, Sergi
AU - Boix, Ester
AU - Prats-Ejarque, Guillem
N1 - Funding Information:
This research work was supported by the Ministerio de Economía y Competitividad (SAF 2015-66007P) co-financed by FEDER funds and Agencia Estatal de Investigación, Spain (PID 2019-106123GB-I00/AEI/10.13039/501100011033), by Fundació La Marató de TV3 (2080310) and AGAUR, Generalitat de Catalunya (2019 LLAV 00002) co-financed by FEDER funds. PF-M was supported by a Juan de la Cierva Incorporación postdoctoral fellowship (Ministerio de Economía y Competitividad) and GP-E is a Margarita Salas postdoctoral researcher (Ministerio de Universidades, Next Generation EU).
Publisher Copyright:
Copyright © 2022 Fernández-Millán, Vázquez-Monteagudo, Boix and Prats-Ejarque.
PY - 2022/9/14
Y1 - 2022/9/14
N2 - Design of novel antibiotics to fight antimicrobial resistance is one of the first global health priorities. Novel protein-based strategies come out as alternative therapies. Based on the structure-function knowledge of the RNase A superfamily we have engineered a chimera that combines RNase 1 highest catalytic activity with RNase 3 unique antipathogen properties. A first construct (RNase 3/1-v1) was successfully designed with a catalytic activity 40-fold higher than RNase 3, but alas in detriment of its anti-pathogenic activity. Next, two new versions of the original chimeric protein were created showing improvement in the antimicrobial activity. Both second generation versions (RNases 3/1-v2 and -v3) incorporated a loop characteristic of RNase 3 (L7), associated to antimicrobial activity. Last, removal of an RNase 1 flexible loop (L1) in the third version enhanced its antimicrobial properties and catalytic efficiency. Here we solved the 3D structures of the three chimeras at atomic resolution by X-ray crystallography. Structural analysis outlined the key functional regions. Prediction by molecular docking of the protein chimera in complex with dinucleotides highlighted the contribution of the C-terminal region to shape the substrate binding cavity and determine the base selectivity and catalytic efficiency. Nonetheless, the structures that incorporated the key features related to RNase 3 antimicrobial activity retained the overall RNase 1 active site conformation together with the essential structural elements for binding to the human ribonuclease inhibitor (RNHI), ensuring non-cytotoxicity. Results will guide us in the design of the best RNase pharmacophore for anti-infective therapies.
AB - Design of novel antibiotics to fight antimicrobial resistance is one of the first global health priorities. Novel protein-based strategies come out as alternative therapies. Based on the structure-function knowledge of the RNase A superfamily we have engineered a chimera that combines RNase 1 highest catalytic activity with RNase 3 unique antipathogen properties. A first construct (RNase 3/1-v1) was successfully designed with a catalytic activity 40-fold higher than RNase 3, but alas in detriment of its anti-pathogenic activity. Next, two new versions of the original chimeric protein were created showing improvement in the antimicrobial activity. Both second generation versions (RNases 3/1-v2 and -v3) incorporated a loop characteristic of RNase 3 (L7), associated to antimicrobial activity. Last, removal of an RNase 1 flexible loop (L1) in the third version enhanced its antimicrobial properties and catalytic efficiency. Here we solved the 3D structures of the three chimeras at atomic resolution by X-ray crystallography. Structural analysis outlined the key functional regions. Prediction by molecular docking of the protein chimera in complex with dinucleotides highlighted the contribution of the C-terminal region to shape the substrate binding cavity and determine the base selectivity and catalytic efficiency. Nonetheless, the structures that incorporated the key features related to RNase 3 antimicrobial activity retained the overall RNase 1 active site conformation together with the essential structural elements for binding to the human ribonuclease inhibitor (RNHI), ensuring non-cytotoxicity. Results will guide us in the design of the best RNase pharmacophore for anti-infective therapies.
KW - antimicrobial protein
KW - base binding sites
KW - catalytic activity
KW - chimera
KW - crystal structure
KW - RNase 1
KW - RNase 3
UR - http://www.scopus.com/inward/record.url?scp=85138903393&partnerID=8YFLogxK
U2 - 10.3389/fmolb.2022.964717
DO - 10.3389/fmolb.2022.964717
M3 - Article
C2 - 36188223
AN - SCOPUS:85138903393
SN - 2296-889X
VL - 9
JO - Frontiers in Molecular Biosciences
JF - Frontiers in Molecular Biosciences
M1 - 964717
ER -