SUPPLEMENTARY MATERIAL FOR:
Solution NMR structure of the SOS response protein YnzC from Bacillus subtilis
James M. Aramini,1 Seema Sharma,1 Yuanpeng J. Huang,1 G. V. T. Swapna,1 Chi Kent Ho,1
Karishma Shetty,1 Kellie Cunningham,1 Li-Chung Ma,1 Li Zhao,1 Leah A. Owens,1 Mei Jiang,1
1
2
1
1
Rong Xiao, Jinfeng Liu, Michael C. Baran, Thomas B. Acton, Burkhard Rost,
2
and Gaetano T. Montelione1,3
1
Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and
Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, U.S.A. and
Northeast Structural Genomics Consortium
2
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY
10032, U.S.A. and Northeast Structural Genomics Consortium
3
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine
and Dentistry of New Jersey, Piscataway, NJ 08854, U.S.A.
Materials and Methods
Full-length and truncated constructs of the ynzC gene from Bacillus subtilis were cloned
into pET21 expression vectors (Novagen) containing a C-terminal affinity tag (LEHHHHH),
yielding the plasmids SR384-21 and SR384-1-46-21. Plasmids were transformed into codon
enhanced BL21 (DE3) pMGK E. coli cells, which were cultured at 37oC in MJ minimal medium1
containing (15NH4)2SO4 and U-13C-glucose as the sole nitrogen and carbon sources. Initial cell
growth was carried out at 37oC and protein expression was induced at 17oC by isopropyl--Dthiogalactopyranoside (IPTG). Expressed proteins were purified using an AKTAexpress (GE
Healthcare) two-step protocol consisting of HisTrap HP affinity chromatography followed
directly by HiLoad 26/60 Superdex 75 gel filtration chromatography. Samples of U-13C,15N
YnzC and YnzC-1-46 for NMR spectroscopy were concentrated by ultracentrifugation to 1.1 to
1.4 mM in 95% H2O/5% D2O solution containing 20 mM MES, 100 mM NaCl, 10 mM DTT, 5
mM CaCl2 at pH 6.5. Sample purity and identity were confirmed by SDS-PAGE, MALDI-TOF
mass spectrometry, and NMR spectroscopy. Both proteins are monomers according to static
light scattering data.
All NMR data were collected at 20oC on Varian INOVA 500 and 600 MHz and Bruker
AVANCE 600 and 800 NMR spectrometers, processed with NMRPipe,2 and visualized using
SPARKY.3 Complete 1H,
13
C, and
15
N resonance assignments for full length B. subtilis YnzC
and truncated YnzC-1-46 were determined using GFT4,5 and conventional6 triple resonance NMR
methods, respectively, and deposited in the BioMagResDB (BMRB accession numbers 7225 and
15476). Stereospecific isopropyl methyl assignments for all Val and Leu residues were deduced
from characteristic cross-peak fine structures in high resolution 2D 1H-13C HSQC spectra of 5%13
C,100%-15N YnzC and YnzC-1-46.7
Resonance assignments were validated using the
Assignment Validation Suite (AVS) software package.8 Three-bond 3J(HN-H) scalar couplings
were determined using the 3D HNHA experiment.9 1H-15N heteronuclear NOEs were measured
with gradient sensitivity-enhanced 2D heteronuclear NOE approaches.10,11
For the full length YnzC structure determination, initial structure calculations were
performed by AutoStructure 2.1.1,12 interfaced with XPLOR-NIH 2.11.2,13 using peak intensities
from 3D
15
N-edited, 3D
13
C-edited aliphatic and 3D
13
C-edited aromatic NOESY spectra (m =
80 ms), as well as 3J(HN-H) data. The final set of NOE-based distance constraints (sum
averaging), dihedral angle constraints derived by HYPER14 within AutoStructure, hydrogen bond
constraints from AutoStructure, C and C chemical shifts, and 3J(HN-H) data were used in a
final simulated annealing refinement in XPLOR-NIH. The folded N-terminal residues (1 to 42)
of the 20 lowest energy structures out of 100 calculated were deposited into the Protein Data
Bank (PDB ID, 2HEP). The truncated YnzC-1-46 structure was calculated using CYANA
2.115,16 supplied with peak intensities from a 3D simultaneous CN NOESY17 (m = 120 ms) and
broad dihedral angle constraints computed by TALOS (
 ± 50°).18 The 20 structures
with lowest target function out of 100 in the final cycle calculated were further were refined by
restrained molecular dynamics in explicit water using CNS 1.2,19,20 using the final NOE derived
distance constraints, TALOS dihedral angle constraints and hydrogen bond constraints from
AutoStructure 2.1.1. The final refined ensemble of 20 structures (excluding the C-terminal His6)
were deposited into the Protein Data Bank (PDB ID, 2JVD).
Structural statistics and global
structure quality factors, including Verify3D,21 ProsaII,22 PROCHECK, 23 and MolProbity24 raw
and statistical Z-scores, were computed using the PSVS 1.3 software package.25
The global
goodness-of-fit of the final structure ensembles with the NOESY peak list data were determined
using the RPF analysis program.26 Ribbons and electrostatic surface potential images of the final
structures were made using MOLMOL 2K.2.27
Figure S1. Static light scattering results for full length (blue) and truncated (1-46; red) YnzC.
Data were collected on a miniDAWN Light Scattering instrument (Wyatt Technology) at = 690
nm and at room temperature on NMR samples of U-13C,15N YnzC and YnzC-1-46 at pH 6.5.
Inset: Plot of molar mass versus elution volume for full length (blue) and truncated (1-46; red)
YnzC. The experimental molecular weights of the two proteins are as follows (expected MW in
parentheses): full length YnzC, 11.3 kDa (10.44 kDa); YnzC-1-46, 7.3 kDa (6.63 kDa).
Figure S2. Overlay of 600 MHz 1H-15N HSQC NMR spectra for full length (blue) and truncated
(1-46; red) YnzC from B. subtilis, obtained at 20oC in pH 6.5 buffer. Assignments for the full
length protein are shown. Side chain Arg N/H peaks aliased in the 15N-dimension of the spectra
are boxed. Inset: Plot of 1HN chemical shift for common assigned residues in full length YnzC
versus YnzC-1-46. Analogous results were obtained for the N, C´, C, C and H assignments
(not shown).
Figure S3.
Figure S3. NMR connectivity map for full length B. subtilis YnzC. Intraresidue (i) and
sequential (s) connectivities for the three-rung assignment strategy28 matching intraresidue and
sequential C [HNCO and HN(CA)CO], C, and C [(4,3)D CC(CO)NHN and (4,3)D
HNNCC] resonances, as well as providing sequential H and H [(4,3)D HC(CO)NHN]
resonances, are shown as horizontal red and yellow lines, respectively.4,5 3J(HN-H) values range
as follows: (o) < 5.5 Hz; ( ) 5.5 ≤ J ≤ 7.5 Hz; (●) > 7.5 Hz. Interresidue NOE connectivities are
shown as thin, medium, and thick black lines, corresponding to weak, medium, and strong NOE
interactions. Bar graphs of the consensus CSI29 and 1H-15N heteronuclear NOE data are shown
in blue.
The secondary structural elements in the experimentally-determined final YnzC
structure (2HEP) and predicted by PROF30 are also shown.
The alpha helices in the final
structure are well defined by the 3J(HN-H) scalar coupling, 1H-15N heteronuclear NOE, and
NOESY patterns, while the unstructured C-terminal region of the protein is indicated by the
reduced 1H-15N heteronuclear NOE values, the CSI data, and the sparse NOESY data.
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