Extended Figure 4. Biophysical characterization of T1232A substitution on the NSD3_SET domain.

a, Kd values with corresponding errors being s.d. are shown for binding studies of NSD3_SET and NSD3_SET-T1232A to recombinant nucleosomes (rNuc) reconstituted on 147bp and 187bp 601 Widom DNA as indicated (determined by microscale thermophoresis (MST)) and the cofactor SAM (determined by ITC) (see Supplementary Table 1; see Methods). Error bars represent mean ± s.d. from three independent experiments. b, The topology of NSD3SET domain segments of Pre-SET (white-gray), SET (cyan), Post-SET (dark gray) and regulatory loop (magenta) marked onto the ribbon representations of existing NSD3SET domain crystal structures (PDB 5UPD, 6CEN). The missing residues in the tip of the regulatory loop, not modelled into the electron densities, are denoted with dotted lines. The zinc(II) ions are depicted as gray spheres, SAM cofactor with sticks. Residues T1232 and V1243 are marked with their side-chains. Bottom panel: Schematic representation of the primary sequence with indicated domains and the location of the T1232A substitution. c, The overlay of the 2D [¹H,¹⁵N] TROSY-HSQC (Transverse Relaxation Optimized SpectroscopY - Heteronuclear Single Quantum Coherence) spectra of 250 μM U-[¹⁵N] NSD3SET wild-type (dark gray) and NSD3_SET-T1232A (magenta) at pH 7.5 and 25°C. The ¹H/¹⁵N backbone amide cross-peaks of T1232 in NSD3_SET wild-type and A1232 of NSD3_SETT1232A mutant are marked. d, The backbone amide chemical shift perturbations (CSP_i = [(Δδ_H,i)²+0.25·(Δδ_N,i)²]^0.5) between NSD3_SET and NSD3_SET-T1232A are mapped onto the ribbon representations of protein structures; left: the NSD3_SET with regulatory loop in closed (PDB 5UPD), center: in open (based on the NSD2_SET crystal structure (PDB 5LSU) modelled with SwissModel) and right: the open conformation with the H3.1 A²⁹-R⁴² substrate docked (see Methods). The T1232 and V1243 residues are marked with their side-chains. Residues are colored as follows: 0.03 ≤ CSP_i < 0.05 (yellow), 0.05 ≤ CSP_i < 0.11 (light orange), 0.11 ≤ CSP_i < 0.75 ppm (orange), CSP_i > 4.60 (red), prolines and residues missing amide assignments are marked gray. e, The overlay of NSD3_SET domains with the regulatory loop in open and closed conformations. The open state is the crystal structure of NSD3_SET, while the closed state is based on the NSD2_SET crystal structure (PDB 5LSU) modelled with SwissModel. The protein sequence graph and coloring as in 4h. f, The projections of 2D TROSY-type heteronuclear {¹H}-¹⁵N nuclear Overhauser effect (NOE) experiments collected for 370 μM U-[¹⁵N] NSD3SET wild-type (left panel) and NSD3_SET-T1232A (right panel) at pH 7.5 and 25°C with several residues marked. (Experimental details in Methods). The higher NOE ratio value the more rigid the N-H vector, meaning less dynamic motion, whereas the lower value means more dynamic, indicative of less restricted motions. The tryptophan imidazole side-chain ¹H/¹⁵N correlations are also detected in this experiment, H^ε1/N^ε1 from W1235 and W1157. g, The observed changes in {¹H}-¹⁵N NOE values (ΔNOE_i) reporting on ps-ns polypeptide main-chain mobility differences between NSD3_SET and NSD3_SET-T1232A are plotted on the 3D static structures of NSD3SET (PDB: 6CEN, left column represented as protein surface, middle column ribbon with H3 peptide, right column ribbon with no substrate). Key features are indicated. The light blue to dark blue indicates the decreased fast dynamics after T1232A substitution, and yellow to red increased dynamics on ps-ns time scale. h, The T1232A substitution enhances the mobility of the regulatory loop. The observed changes in heteronuclear {¹H}-¹⁵N nuclear Overhauser effect (NOE) mapped on the ribbon representations of the open and closed states of the regulatory loop within the NSD3_SET and in Figure 2e. The structures are the same as in (e). i, Methylation assays as in Figure 2a with rNuc(187bp) and the indicated NSD3 variants.