SUPPLEMENTARY MATERIAL FOR:
Solution NMR Structure of Dsy0195 Homodimer from Desulfitobacterium hafniense: First Structure Representative of the YabP Domain Family of Proteins Involved in Spore Coat Assembly
Yunhuang Yang,1,2 Theresa A. Ramelot,1,2 John R. Cort,2,3 Huang Wang,2,4 Colleen Ciccosanti,2,4 Mei Jiang,2,4 Haleema Janjua,2,4 Thomas B. Acton,2,4 Rong Xiao,2,4 John K. Everett,2,4 Gaetano T. Montelione,2,4,5 Michael A. Kennedy1,2*
1Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056
2Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352
4Department of Molecular Biology and Biochemistry, Center for Advanced Biotechnology and Medicine, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854
5Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854
Materials and Methods
(1) Protein constructs, expression and purification
The truncated Dsy0195 (residues 21-82) of Desulfitobacterium hafniense strain Y51 (UniProt/TrEMBL ID, Q251Q8_DESHY; NESG ID, DhR8C) was cloned into pET21_NESG containing a C-terminal affinity tag (LEHHHHHH) to yield the plasmid DhR8C-21.3. The DhR8C-21.3 plasmid was transformed into codon-enhanced BL21 (DE3) pMGK Escherichia coli cells, and cultured in MJ9 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 1 mM isopropyl-b-D-thiogalactopyranoside at mid-log phase growth. Expressed proteins were purified using an ÄKTAxpress™ (GE Healthcare) two-step protocol consisting of HisTrap HP affinity chromatography followed directly by HiLoad 26/60 Superdex 75 gel filtration chromatography. The final yield of purified isotopically-enriched Dsy0195 was approximately 76.7 mg/L of culture. Samples of [U-13C,15N]- and [U-5%-13C,100%-15N]-Dsy0195 for NMR spectroscopy were concentrated by centrifugation to 0.9 to 1.1 mM in 90% H2O/10% 2H2O solution containing 20 mM ammonium acetate, 100 mM NaCl, 10 mM DTT, 5 mM CaCl2 at pH 4.5. Sample purity and molecular mass were confirmed by SDS-PAGE and MALDI-TOF mass spectrometry (MALDI-TOF mass of [U-13C, 15N]-Dsy0195 (Da): experimental, 8,438; expected, 8,447).
(2) Gel filtration chromatography
Analytical gel filtration with static light scattering detection was carried out for Dsy0195 on a miniDAWN (TREOS) Light Scattering instrument (Wyatt Technology) at l= 690 nm and at 30oC on an NMR sample of [U-13C,15N]-Dsy0195 at 20mM ammonium acetate (pH 4.5), 100mM NaCl, 10mM DTT, 5 mM CaCl2 and 0.02% sodium azide. The resulting experimental molecular weight of 16.9 kDa indicated its dimeric state under NMR sample buffer conditions.
(3) Spin labeling sample preparation for PRE and DEER experiments
Two single cysteine mutants of Dsy0195 (S36C and S52C) were generated by using the QuikChange site-directed mutagenesis kit (Stratagene). (1-Oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate (MTSL) was used to spin-label free cysteine in the S36C and S52C mutants in the NMR buffer adjusted to 10% glycerol for better solubility instead of 10mM DTT. Free sulfphydryl groups were modified over 48 hours at room temperature with 3-5 fold molar excess MTSL dissolved in acetone. Free MTSL was removed by size exclusion chromatography prior to PRE or DEER measurements. For PRE experiments, 1:1 molar ratio of 15N-labeled wt/mutant S36C (or S52C) samples were prepared. 2D 15N-HSQC spectra were recorded on the Bruker AVANCE 850 MHz NMR spectrometer at 20oC. Following the NMR analysis in the oxidized form, samples were reduced by adding a 2 to 3 fold molar excess of 200 mM ascorbic acid. The inter-monomer distances from PRE broadening were extracted followed the strategy proposed by Rumpel et al2. For DEER experiments, about 0.25 mM MTSL spin labeled Dsy0195 was disolved in 20 mM ammonium acetate, 100 mM NaCl, and 5 mM CaCl2 (pH 4.5) diluted to a final concentration of 30% (w/w) glycerol. The DEER experiment was carried on a Bruker ELEXSYS E580 pulsed EPR instrument at 80 K3.
(4) NMR spectroscopy
15N T1 and T2 relaxation measurements were made using 1D 15N T1 and T2 (CPMG) relaxation experiments on Varian INOVA 600 MHz NMR spectrometer at 25oC. T1 spectra were acquired with delays, T = 100, 200, 300, 400, 700, 1000, 1500, and 2000 ms, and a relaxation delay of 3 s. T2 spectra were acquired with CPMG delays, T = 10, 30, 50, 70, 110, 130, 150, and 170 ms, and with a relaxation delay of 1.5 s. 15N T1 and T2 values were extracted by plotting the decay of integrated 1HN intensity between d ≈ 8.5 to 10.5 ppm and fitting the curves with standard exponential equations. The tc was calculated from the 15N T1/T2 ratio using the following approximation of literature relaxation equations4:
(1)
where nN is the resonance frequency of 15N in Hz. Using this approach, we obtain a tc of 12.2 ns for [U-13C,15N]-Dsy0195, which is consistent with a dimer (expected MW = 16.9 kDa, including C-terminal affinity tag).
All NMR data for resonance assignment and structure determination were collected at 20oC on Varian INOVA 600 and Bruker AVANCE III 850 MHz NMR spectrometers. The edited/filtered 13C-edited NOESY spectrum5 was collected on 1:1 molar ratio of [U-13C, 15N]: nature abundance Dsy0195 sample for intramonomer NOEs detection.
(5) Structure calculation of Dsy0195 monomer and homodimer
All NMR data were processed with NMRPipe6 and visualized using SPARKY.7 All spectra were referenced to internal DSS. Complete 1H, 13C, and 15N resonance assignments for Dsy0195 were determined using conventional triple resonance NMR methods. Backbone resonance assignments were made by AutoAssign 2.4.08,9 using peak lists from 2D 1H-15N HSQC and 3D HNCO, HNCA, HNCO(CA), HNCACB and CACB(CO)NH spectra. One small segment of Dsy0195 ranging from residue 60 to 64 (EILLE) is dynamically flexible and those residues were completely missed in the NMR spectra and their chemical shifts were not capable of being assigned. Side chain assignment was completed manually using 3D HBHA(CACO)NH, HCCH-COSY, HCCH-TOCSY and (H)CCH-TOCSY experiments. 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 [U-5%-13C,100%-15N]-Dsy019510.
Dihedral angle constraints were computed by TALOS11 (f ± 30°; y ± 30°) for ordered residues with confidence scores of 10. Hydrogen bond constraints were derived from AutoStructure 2.1.112. The NMR structure of Dsy0195 monomer was calculated using CYANA 2.113 supplied with peak intensities from 3D 13C-edited NOESY, 15N-edited NOESY, and 4D 13C-13C-HMQC-NOESY-HMQC spectra with NOE mixing time (tm) 70 ms, together with dihedral angle and hydrogen bond constraints. NMR structure quality analyses were performed using the PSVS 1.414 and RPF15 software packages.
The final Dsy0195 homodimer structure was determined in two different ways, based on inter-monomer distance restraints derived from (a) edited/filtered 13C-edited NOESY spectrum, or (b) from PRE2 and DEER16 measurements of spin labeled Dsy0195 samples. The 20 structures with lowest target function out of 150 structures in the final cycle calculation were further refined by CNS 1.2 with explicit water. The final ensemble of 20 models of Dsy0195 homodimer structure were deposited into the Protein Data Bank (PDB ID: (a) 2KS0 and (b) 2KYI), respectively. Structural statistics and global structure quality factors, including Verify3D17, ProsaII18, PROCHECK19, and MolProbity20,21 raw and statistical Z-scores, were computed using the PSVS 1.4 software package. The global goodness-of-fit of the final structure ensemble with the NOESY peak list data and resonance assignments was determined using the RPF analysis program.
Supplementary Figure S1.
Supplementary Figure S1. Analytical gel filtration with static light scattering detection for an NMR sample of 13C/15N Dsy0195. Inset: Plot of molar mass versus elution time. Based on the monomeric molecular weight (MW) including affinity tag is 8.4 kDa, the resulting experimental molecular weight of 16.9 kDa indicated a dimeric state under the NMR buffer conditions.
Supplementary Figure S2.
Supplementary Figure S2. 1D 15N T1 and T2 relaxation data for [U-13C,15N]-Dsy0195. (Top): 15N T1 and T2 values were extracted by plotting the decay of integrated 1HN intensity between d ≈ 8.5 to 10.5 ppm and fitting the curves with standard exponential equations. (Bottom): Plot of rotational correlation time, tc (ns), versus protein molecular weight (kDa) for known monomeric NESG targets of ranging size (taking into account isotope enrichment as well as affinity tags in the sequence). 15N T1/T2 data for all monomeric proteins used for the tc vs. MW plot were obtained on the same Varian INOVA 600 MHz spectrometer at 25 oC, and tc was analyzed as described in Materials and Methods section. tc of 12.2 ns for [U-13C,15N]-Dsy0195 is consistent with a dimer (expected MW = 16.9 kDa, including C-terminal affinity tag).
Supplementary Figure S3.
Supplementary Figure S3. Two dimensional 1H-15N HSQC spectrum of about 1 mM [U-13C,15N]-Dsy0195 in 90% H2O/10% D2O solution containing 20 mM ammonium acetate, 100 mM NaCl, 10 mM DTT, 5 mM CaCl2 at pH 4.5 collected at 20oC on a Varian Inova 600 MHz NMR spectrometer. Backbone resonance assignments are labeled with one-letter amino acid codes followed by their sequence numbers. Assigned side chain NHe resonances of Arg (aliased) and side chain NH2 resonances of Asn and Gln are also indicated.
Supplementary Figure S4.
Supplementary Figure S4. Motifs and sites analysis on the lowest energy conformer of the final NMR ensemble of Dsy0195 homodimer (PDB ID: 2KYI) (http://www.ebi.ac.uk/pdbe-site/pdbemotif/). One 22-residue segment ranging from residue 44 to 65 (L44-(x)6-L51-(x)6-L58-(x)6-L65) shows the sequence character of a leucine zipper motif.
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