Fig. 3. The frHMGB1•CXCL12 heterocomplex forms via fuzzy interactions.

a Superposition of selected regions of ¹H-¹⁵N HSQC spectra of 0.1 mM ¹⁵N frHMGB1 (corresponding to spy residues W48, T76, I78, A93, I158) without (black) and with 0.2 mM CXCL12 (red) and 0.1 mM ¹⁵N frHMGB1-TL (blue). CXCL12 partially competes with intramolecular frHMGB1 interactions and specific amide resonances move (arrow) towards the chemical shift of the corresponding amide in the tailless construct. b ¹H-¹⁵N HSQC spectra of frHMGB1 (0.1 mM) without (black) and with 0.2 mM CXCL12 (red), and with subsequent addition of 0.1 mM Ac-pep (green). Grey shadowed regions highlight resonances disappearing and reappearing upon addition of CXCL12 and Ac-pep, respectively. Bar graphs showing (c) residue-specific chemical shift perturbation (CSP) and (d) peak intensity ratios (I/I₀) of ¹⁵N-labeled frHMGB1 (0.1 mM) upon addition of CXCL12 (1:1). Residues with CSP > avg + σ₀ (corrected standard deviation, blue line) and I/I₀ <avg - SD (standard deviation, blue line) are colored (blue) and (e) mapped on frHMGB1 (grey surface, Aphafold2 model AF- P63159). Bar graph showing (f) residue-specific CSPs and (g) I/I₀ of ¹⁵N-labeled frHMGB1-TL (0.1 mM) upon addition of CXCL12 (1:1). Residues with CSP > avg + σ₀ (magenta line) and I/I₀ <avg - SD (magenta line) are colored in magenta and (h) mapped on frHMGB1-TL (grey surface, pdb code: 2YRQ). In the bar-graphs α-helices are schematically represented on the top, missing residues are either prolines, or superimposed residues of the acidic IDR or absent because of exchange with the solvent, the dashed black line indicates the peak intensity decrease due to the titration dilution effect. Source data are provided as a Source Data file.