NMR measurements

Proteins were prepared in 100 mM potassium phosphate, 1 mM EDTA, pH 6.5. Pooled and concentrated fractions were dialyzed in gel filtration buffer plus 0.03% w/v sodium azide. Chemical shift assignments were made using standard protein NMR methodology. Preliminary HSQC-TROSY experiments were conducted on a 600 MHz Bruker spectrometer (Billerica, MA) at the Biomolecular NMR Core at the University of Chicago. Final NMR experiments were executed at the University of Minnesota NMR Center on a Bruker Ascend 850 MHz spectrometer equipped with a TCl CryoProbe, with the sample temperature held constant at 308 K. The HN(CA)CO experiment was performed on a Varian 600 MHz spectrometer equipped with a ¹³C enhanced cold HCN Z-gradient probe. HNCA, HN(CO)CA, HNCO, HN(CA)CO CBCA(CO)NH, and CBCANH experiments were used to assign the backbone atom resonances for residues 883 through 949. Likewise, HCCCONH, HBCBCGCDHD, HCCH-TOCSY, and HCCH-COSY spectra were used to assign the side-chain ¹H and ¹³C resonances for residues 884 through 949, all the M10^short-GCN4 residues. Distance restraints were obtained from 3D ¹⁵N-NOESY (200 ms mixing time) and 3D ¹³C-NOESY (200 ms mixing time) experiments. Data were processed using NMRPipe (17) and analyzed using the CCPNMR software suite (18).

Nuclear Overhauser effect (NOE) peak volumes were calibrated using the average NOE volume from geminal Hβ atoms and classified into the following distance bins: short (1.8–3.7), medium (1.8–5.0), and long (1.8–6.0). Dihedral constraints were calculated using TALOS+ software (19). NOE distance constraints were imported into XPLOR-NIH and used for NMR structure calculation (20). We generated a composite containing 10 lowest energy structures from 100 calculated structures. We used The Protein Structure Validation Suite (PSVS), version 1.5 (21) to validate structures, and VMD and VMD-Xplor to generate molecular representations (22). The SBGrid Consortium provided software binaries (23).