Nucleolin uses its RBD2 domain to recognize long-loop G-Quadruplexes from human minisatellite CEB25

ABSTRACT: We investigated herein the interaction between nucleolin (NCL) and a set of G4 sequences derived from the CEB25 human minisatellite which adopt a parallel topology while differing by the length of the central loop (from 9nt to1nt). It is revealed that NCL strongly binds to long-loop (9-5 nt) G4 whilst interacting weakly with the shorter variants (loop < 3nt). Photocrosslinking experiments using 5-bromouracil (BrdU) modified sequences further confirmed the loop-length dependency thereby indicating that the CEB25-WT (9nt) is the best G4 substrate. Quantitative proteomic analysis (LC-MS/MS) of the photocrosslinking product(s) obtained with NCL bound to this sequence enabled the identification of one contact site within the 9nt loop. The protein fragment identified is located in the helix of the RBD2 domain of NCL, shedding light on the role of this structural element in the G4-loop recognition. Then, the ability of a panel of benchmark G4 ligands to prevent the NCL/G4 interaction was explored. It was found that only the most potent ligand PhenDC3 is able to inhibit NCL binding, thereby suggesting that the terminal guanine quartet is also a strong determinant of G4 recognition, putatively through interaction with the RGG domain. This study puts forward the molecular mechanism by which NCL recognizes G4-containing long loops and leads to propose a model implying a concerted action of RBD2 and RGG domains to achieve specific G4 recognition via a dual loop-quartet interaction.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Valentin SABATET  

LAB HEAD: Damarys Loew

PROVIDER: PXD016529 | Pride | 2020-03-19

REPOSITORIES: Pride