Interactions between a Heparin Trisaccharide Library and FGF-1 Analyzed by NMR Methods

Abstract

FGF-1 is a potent mitogen that, by interacting simultaneously with Heparan Sulfate Glycosaminoglycan HSGAG and the extracellular domains of its membrane receptor (FGFR), generates an intracellular signal that finally leads to cell division. The overall structure of the ternary complex Heparin:FGF-1:FGFR has been finally elucidated after some controversy and the interactions within the ternary complex have been deeply described. However, since the structure of the ternary complex was described, not much attention has been given to the molecular basis of the interaction between FGF-1 and the HSGAG. It is known that within the complex, the carbohydrate maintains the same helical structure of free heparin that leads to sulfate groups directed towards opposite directions along the molecular axis. The precise role of single individual interactions remains unclear, as sliding and/or rotating of the saccharide along the binding pocket are possibilities difficult to discard. The HSGAG binding pocket can be subdivided into two regions, the main one can accommodate a trisaccharide, while the other binds a disaccharide. We have studied and analyzed the interaction between FGF-1 and a library of trisaccharides by STD-NMR and selective longitudinal relaxation rates. The library of trisaccharides corresponds to the heparin backbone and it has been designed to interact with the main subsite of the protein.

Boosting the NMR Assignment of Carbohydrates with Clean In‐Phase Correlation Experiments

“…The performance of the basic CLIP-COSY and the proposed relayed experiments of Figure 1w as tested on the novel, bioisosterics ulfonica cid heparin analogue trisaccharide [15] 1 (Scheme 1) which has potentialb iological significance as an anticoagulant. [16] As mentioned before,o ptimal signal-to-noise ratios in the single-and double-relayeds pectra were attained by setting the duration of the perfect-echom ixing (D)t o2 0-25 ms, which is an appropriate compromise for all protons of 1 displaying couplings from 2t o1 4Hz( see Figure S1 for experimental data demonstrating the echo-mixing time (D)d ependence of representative CLIP-COSY peaks of 1). In general,t he corresponding mixing time for the CLIP-COSY experiment and its relayed variants should be chosen wisely for efficient coherence transfer.C onsidering the transfer of spin 1t os pin 2i na Figure 1.…”

“…The performance of the basic CLIP‐COSY and the proposed relayed experiments of Figure was tested on the novel, bioisosteric sulfonic acid heparin analogue trisaccharide 1 (Scheme ) which has potential biological significance as an anticoagulant …”

“…In the case of double relay (n = 3), f 1 = (x) 8 ,(Àx) 16 ,(x) 8 ; f 7 = (y) 4 ,(Ày) 4 in the third perfect-echo element is ap-…”

The Use of NMR to Study Transient Carbohydrate—Protein Interactions

“…T 1sel and T 2 also depend on correlation times and in principle they can be used to study association processes for fast equilibrium between ligand and receptor, observing the ligand signals at low concentration of the receptor. These values can be used for the analysis of binding constants by performing a titration (García-Jiménez et al, 2017 ). A series of T 1sel /T 2 values are obtained at different ratios of ligand to receptor, and by fitting these to the variation in the ligand/receptor ratio, K D or IC 50 can be extracted.…”

Discovery of exolytic heparinases and their catalytic mechanism and potential application

“…Structurally, Hepase I folds into a β-jelly roll-type structure and prefers to cleave the α-1,4-linkages connected to IdoA2S/GlcA2S residues 18,19 . In contrast, Hepase III is composed of an N-terminal (α/α) 5 barrel domain and a C-terminal antiparallel β-sandwich domain and prefers to cleave the α-1,4-linkages connected to GlcA/IdoA residues 18,20 . Hepase II adopts a topology similar to Hepase III and has no selectivity for certain GlcA/IdoA or IdoA2S/GlcA2S structures 18,21,22 .…”