Volume 113, Issue 12, Pages 2609-2620 (December 2017) Nanoscale Assembly of High-Mobility Group AT-Hook 2 Protein with DNA Replication Fork  Natalie Krahn, Markus Meier, Vu To, Evan P. Booy, Kevin McEleney, Joe D. O’Neil, Sean A. McKenna, Trushar R. Patel, Jörg Stetefeld  Biophysical Journal  Volume 113, Issue 12, Pages 2609-2620 (December 2017) DOI: 10.1016/j.bpj.2017.10.026 Copyright © 2017 Biophysical Society Terms and Conditions

Figure 1 (A) Given here is a schematic of the amino acid sequence of human HMGA2 highlighting the AT-hook domains. (B) Given here is a Western blot of HMGA2 knockdown at 48 h post transfection using an anti-Tubulin (control) and anti-HMGA2 antibody. (C) The effect of HMGA2 knockdown on cell viability was monitored for HEK293T cells at 24-h intervals over 96 h. A significant loss in viability was observed upon transfection with the HMGA2 siRNA. Data points are shown as an average of eight measurements with error bars corresponding to the SD. (D) Shown here is the Coomassie-stained SDS-PAGE of purified HMGA2. To see this figure in color, go online. Biophysical Journal 2017 113, 2609-2620DOI: (10.1016/j.bpj.2017.10.026) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 2 (A) Shown here are elution profiles of HMGA2 (coral), RF (teal), and complex (sea green) from the Superdex 75 10/300 GL, HiLoad 16/600 Superdex 75, and Superdex 75 10/300 GL SEC columns, respectively, indicating purity of both individual components as well as the complex. (B) Given here is EMSA indicating slower migration of RF (200 nM) upon interaction with HMGA2 (0–3 μM). (C) Shown here is the binding profile of HMGA2 with RF fit to a modified Hill equation. Data points are determined using densitometry and displayed as the average of three independent measurements (740 ± 50 nM). To see this figure in color, go online. Biophysical Journal 2017 113, 2609-2620DOI: (10.1016/j.bpj.2017.10.026) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 3 (A) Given here is the hydrodynamic radius (Rh) distribution and (C) sedimentation coefficient (s20,w) distribution of HMGA2 (coral), RF (cyan), and complex (sea green) at 1 mg/mL, suggesting monodisperse samples. (B) The Rh for HMGA2 and RF is plotted at multiple concentrations (1–10 mg/mL, bottom x axis) and for the complex between 0.2 and 1 mg/mL (top x axis). The vertical error bars indicate the SD from five measurements. (D) The Svedberg coefficient in water (s20,w) was plotted at multiple concentrations (0.2–2 mg/mL) for each sample with vertical error bars indicative of the SE from multiple scans. This plot presents the concentration independence of all samples under the experimental conditions. To see this figure in color, go online. Biophysical Journal 2017 113, 2609-2620DOI: (10.1016/j.bpj.2017.10.026) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 4 The entire sequence of HMGA2 is predicted to be disordered according to the program RONN (52). To see this figure in color, go online. Biophysical Journal 2017 113, 2609-2620DOI: (10.1016/j.bpj.2017.10.026) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 5 (A) Given here is the far-UV CD spectra of HMGA2, RF, and complex displayed as an average of three measurements indicating disordered HMGA2 and confirmation of RF formation. A reference RF (56) (dotted black) spectrum was overlaid onto the experimental RF (teal) for comparison. (B) Shown here is the 15N HSQC spectral overlay of free HMGA2 (coral) with HMGA2 in complex with RF (green), suggesting a conformational change of HMGA2 upon interaction with RF. (C) Given here is merged SAXS data of HMGA2 (coral), RF (teal), and complex (sea green) obtained from multiple concentrations. The corresponding (D) Kratky plot suggests that HMGA2 (coral) is a disordered protein and that RF (teal) is structured (upper panel). Theoretical Kratky analysis of the complex (purple) is distinctly different from the experimental analysis (sea green), suggesting that upon interaction with RF, HMGA2 undergoes a conformational change (lower panel). (E) Given here is the P(r) function of HMGA2, RF, and complex (same color scheme as C and D) outlining the multidomain nature of the molecules and their Dmax. To see this figure in color, go online. Biophysical Journal 2017 113, 2609-2620DOI: (10.1016/j.bpj.2017.10.026) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 6 Shown here are low-resolution structures of (A) RF (extended shape) and (B) complex determined by DAMMIN. (C) Modeling by MONSA distinguishes HMGA2 (coral) from RF (blue) in the complex, suggesting that HMGA2 interacts with RF in a side-by-side orientation. To see this figure in color, go online. Biophysical Journal 2017 113, 2609-2620DOI: (10.1016/j.bpj.2017.10.026) Copyright © 2017 Biophysical Society Terms and Conditions