Manganese Superoxide Dismutase from Human Pathogen Clostridium Difficile

Electronic Supporting Information

Manganese superoxide dismutase from human pathogen Clostridium difficile

Wei Li, Hongfei Wang, Cheng Lei, Tianlei Yingand Xiangshi Tan*

Wei Li

Institutes of Biomedical Sciences, Fudan University

Shanghai 200433, China

Cheng Lei, Tianlei Ying

Department of Chemistry, Fudan University

Shanghai 200433, China

Hongfei Wang

State Key Laboratory of Quantum Optics and Quantum Optics Devices, Shanxi

University,Taiyuan 030006, China

X Tan ()

Department of Chemistry & Institutes of Biomedical Sciences,

Fudan University, Shanghai 200433, China

E-mail:

Fax: +86 21 65641740

1. The subcloning strategy and purification of SODcd

TheSODcd gene was inserted to the linear pMAL-c2x expression vector and the protein purity was confirmed by SDS-PAGE. For the subunit assembly determination, we performed the gel filtration chromatography on a HiLoad™ 16/60 Superdex™ 200 high-resolution column. The column was equilibrated with 20 mM Tris–HCl, 150 mM NaCl (pH 7.4) and was calibrated with a molecular mass marker kit (12 to 200 kDa; Sigma) containing blue dextran (2,000 kDa), alcohol dehydrogenase (150 kDa), albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa). MBP from (43 kDa) was also used as a column standard.

Fig. S1:SDS-PAGE analysis of MnSODcd after cleavage of MBP tag andthesuperdex S200 gel filtration profile of MnSODcd as a dimer.

1. MS spectrometryof MnSODcd

MnSODcd band in fresh CCB-stained gel was excised and plated into a 96-well microtitre plate. Excised slices were firstly destained twice with 60 μl of 50 mM NH4HCO3 and 50% acetonitrile and then dried twice with 60μl of acetonitrile. Afterwards, the dried pieces of gels were incubated in ice-cold digestion solution (typsin 12.5 ng/μl and 20mM NH4HCO3) for 20 min and then transferred into a 37°C incubator for digestion overnight. Finally peptides in the supernatant were collected after extraction twice with 60μl extract solution (5% formic acid in 50% acetonitrile). The peptide solution described above was dried under the protection of N2. The 0.8 μl matrix solution (5mg/ml α-cyano-4-hydroxy-cinnamic acid diluted in 0.1％TFA, 50％ACN) was pipetted to dissolve it. Then the mixture was spotted on a MALDI target plate (AB SCIEX). MS analysis of peptide was performed on an AB SCIEX 5800 TOF/TOF. The UV laser was operated at a 400 Hz repetition rate with wavelength of 355 nm. The accelerated voltage was operated at 20 kV, and mass resolution was maximized at 1600 Da. Myoglobin digested with trypsin was used to calibrate the mass instrument with internal calibration mode. All acquired spectra of samples were processed using TOF/TOF ExplorerTM Software (AB SCIEX) (Janiszewski et al. 2012) in a default mode. The data were searched by GPS Explorer (V3.6) with the search engine MASCOT(2.3) (Sharifah et al. 2011). The search parameters were as follows: the databaseNCBInr 20100724 (11505486 sequences; 3925745078 residues), trypsin digest with one missing cleavage，MS tolerance was set at 100 ppm，MS/MS tolerance of 0.6 Da. Protein with scores greater than 75 were significant (P<0.05).

Fig. S2: The MS/MS spectrum of MnSODcd. The upper two MS spectrum showedthe highest intensity peaks at M/Z = 922.9458 and M/Z = 2758.8254, representing the peptide sequences of 143KFKQEFQKS149 coming from the middle helix α5 and 207RRNEYIDNWFNVVNWNGALENYKN228 located in the C-terminal of SODcd, respectively. TheMascot search resultsconfirmed the authenticity of theMnSODcd.

1. The superimposing structures of different SODs

Crystal structures of SODs from C. difficile,H. sapiens, E.coli and P. gingivalis, respectively, were compared.

Fig. S3: Thestructural alignment of different SODs. Panel A, the overall structural comparisons among the structures of C. difficile MnSOD (green, PDB ID 4JZG),H. sapiens Mn-specific SOD (purple, 1LUV), E.coli Fe-speific SOD (yellow, 1ISB) and P. gingivalis cambialistic SOD (blue,1QNN). Panel B, the alignment of the active site residues from the first coordination sphere and the second coordination sphere. Gly191 and Phe114 are key discriminating residues to determinate the type of Mn/Fe SOD.

1. In silico molecular docking 2-methoxyestradiolinto MnSODcd.

The docking procedure Flexible Docking (Jones et al. 1997) integrated in Discovery Studio (Accelrys company) was used. All the modules are pipelined to work coordinately. MnSOD structure from this study (PDB ID: 4JZG) was used as the initial model and the irrelevant waters were removed. Firstly,conformations of 2-ME were generated using CatConf /CAESAR which have 230 enantiomers used as the training set. The protein was added hydrogen (Jones et al. 1997). The excess charge was neutralized by the counter-ion pairs of NaCl then the structures were validated using molecular dynamics under full solvation based on CHARMM force field (Guvench and MacKerell 2008). Over a 10 ns time course, no major rearrangements were observed. Then 2-methoxyestradiolwas docked flexibly into MnSODcd surface using the grid surface searching methods. Then, Libdock (Rao et al. 2007) was used to select hotspot poses following modifying side chain with ChiRotor (Spassov et al. 2007). The last step is annealing/energy minimization for ligand pose by CDOCKER (Wu et al. 2003). The conformations having the highest score (based on LigScore1, LigScore2, PLP1, PLP2, Jain, PMF scoring function) (Krovat and Langer 2004) were choose for analyzing the interaction models of 2-ME and MnSODcd.

Fig. S4. Models of 2-methoxyestradiolbeing docked into MnSODcd. Panel A, one binding model with 2-methoxyestradiollocated at the concave of the threeβsheets. Panel B,the second model for 2-methoxyestradiol tobe located at the dimer interface cleft of MnSODcd. The relevant residues and 2-methoxyestradiolare shown in sticks model and protein peptides are represented in cartoon model. This picture was prepared by PyMOL.

Table S1. The Ligandfit score ranking of selected 10 poses in different locations of MnSODcd

1. The primary sequence alignment of MnSODs from different species.

Fig. S5. The sequence alignment of C. difficile, H. sapiens, S. cerevisiae and D. radiodurans MnSOD.The secondary structures according to the crystal of D. radiodurans MnSOD are indicated above the sequence. The identical residues between these SODs are highlighted with red shadow and the conservative residues are included into the blue box with the gaps shown by dot. The blue triangles point the metal coordination residues

References

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