Intramolecular binding mode of the C-terminus of E. coli

Transcription

1 Supporting Information Intramolecular binding mode of the C-terminus of E. coli single-stranded DNA binding protein (SSB) determined by nuclear magnetic resonance spectroscopy Dmitry Shishmarev, Yao Wang, Claire E. Mason, Xun-Cheng Su, Aaron J. Oakley, Bim Graham, Thomas Huber, Nicholas E. Dixon and Gottfried Otting S1

2 Table S1. Long-range NOEs observed in SSB at ph LeuH γ 78TyrH δ 112LeuH δ a δ 78TyrH 112LeuH δ a βb 78TyrH 111MetH γ a δa 71LeuH 111MetH ε 70TyrH ε 111MetH ε 70TyrH δ 110GlnH N 79IleH γ 2 110GlnH N 79IleH δ1 110GlnH γ a ε 78TyrH 110GlnH β a ε 78TyrH 109MetH γ a γ2 79IleH 109MetH β a γa 77ValH 109MetH ε 77ValH γ a 109MetH ε 66ValH γ a 109MetH ε 67AlaH α 109MetH ε 63LeuH α 109MetH α 57ValH γ a 108ThrH N 79IleH γ 2 108ThrH γ 2 ε 78TyrH 108ThrH γ 2 δ 78TyrH 104AsnH α 60PheH β a 103ValH γ a αa 81GlyH 103ValH N 79IleH γ 2 102ValH γ a ε 60PheH 102ValH γ a δ 60PheH 102ValH β 60PheH δ 102ValH α 58ValH γ a 102ValH γ a γa 58ValH 102ValH γ a β 58ValH 101ValH γ b 84ArgH N 101ValH γ b 83LeuH N 101ValH γ b α 83LeuH 101ValH γ b αa 81GlyH 101ValH γ a αa 81GlyH 101ValH γ b 58ValH N 101ValH γ a δ2 55HisH 101ValH γ b δ2 55HisH 101ValH γ b δa 10LeuH 97TyrH δ 87LysH γ b 97TyrH δ 87LysH γ a 97TyrH ε 87LysH γ a 97TyrH δ 87LysH β a 97TyrH δ 85ThrH γ 2 97TyrH ε 85ThrH γ 2 96ArgH β a ε3 88TrpH 96ArgH β b ε1 88TrpH 96ArgH β b ε3 88TrpH 95AspH α 89ThrH γ 2 93GlyH α b γ2 89ThrH 93GlyH α a γ2 89ThrH 80GluH α 9IleH γ 2 80GluH α 9IleH β 79IleH γ 2 δa 10LeuH 79IleH β 10LeuH δ b 78TyrH δ 11ValH α 78TyrH δ 11ValH γ b 78TyrH ε 11ValH γ b 78TyrH ε 11ValH γ a 78TyrH ε 11ValH β 78TyrH δ 11ValH β 78TyrH ε 9IleH δ1 78TyrH ε 9IleH γ 2 78TyrH δ 9IleH γ 2 78TyrH β b γ2 9IleH 77ValH γ b δb 10LeuH 76GlnH α 13AsnH α 76GlnH β a α 13AsnH 73LysH α 16GlnH N 73LysH α 14LeuH δ a 73LysH α 14LeuH γ 70TyrH δ 66ValH α 70TyrH ε 66ValH α 70TyrH ε 66ValH γ a 70TyrH ε 66ValH γ b 70TyrH δ 66ValH γ a 70TyrH δ 66ValH γ b 70TyrH ε 66ValH β 70TyrH δ 66ValH β 70TyrH ε 19GluH γ a 70TyrH δ 10LeuH β b 60PheH α 29ValH γ b 60PheH δ 29ValH γ b 60PheH δ 29ValH γ a 60PheH ε 29ValH γ a 60PheH δ 29ValH γ b 60PheH ε 29ValH γ b 60PheH ζ 29ValH γ b 60PheH ε 23MetH ε 60PheH δ 23MetH ε 58ValH γ a α 31AsnH 57ValH γ a δa 10LeuH 55HisH δ 2 δb 10LeuH 55HisH δ 2 δa 10LeuH 55HisH δ 2 βb 10LeuH 55HisH ε 1 βb 10LeuH 55HisH ε 1 δb 10LeuH S2

3 55HisH ε 1 δa 10LeuH 55HisH β b δa 10LeuH 55HisH β a δa 10LeuH 55HisH β b δb 10LeuH 55HisH β a δb 10LeuH 54TrpH ζ 3 β 35AlaH 54TrpH ε 3 β 35AlaH 54TrpH ζ 3 α 35AlaH 54TrpH ε 3 β 35AlaH 54TrpH ζ 2 β 35AlaH 54TrpH η 2 β 35AlaH 54TrpH ε 1 β 35AlaH 54TrpH ε 3 β 33ThrH 54TrpH ε 3 γ2 33ThrH 54TrpH η 2 γ2 33ThrH 54TrpH ε 3 γb 20ValH 54TrpH ζ 3 αb 15GlyH 54TrpH ζ 3 αa 15GlyH 54TrpH η 2 αb 15GlyH 51GlnH β a η2 40TrpH 36ThrH γ 2 δb 10LeuH 35AlaH β 13AsnH β a 35AlaH β 13AsnH N 35AlaH β 12GlyH α b 32IleH γ 2 19GluH N 32IleH γ 2 βa 18ProH 30AlaH β 20ValH N 29ValH γ b ε 23MetH 29ValH γ a ε 23MetH 29ValH γ b γa 23MetH 29ValH γ a γb 23MetH 28AlaH N 23MetH ε 28AlaH β 22TyrH δ 28AlaH β 22TyrH ε 28AlaH α 22TyrH δ 28AlaH α 22TyrH ε Letters a and b in superscripts identify individual 1 H resonances of CH 2 groups, or individual CH 3 groups in isopropyl groups, using a for the more shielded resonance. All NOEs were suggested by CCPNMR Analysis (30) based on the assigned chemical shifts without reference to the 3D structure of the protein. No NOEs were observed that were not in agreement with the structure of SSB observed in the crystal structure 1EYG (9). S3

4 Table S2. PCSs measured for backbone amide protons of SSB with C1-lanthanide tag residue Tm 3 Tb 3 S R G V N K V I L V G N L G D E G V I L A T S E S W R D K A G E M K E Q H residue Tm 3 Tb 3 R V K G S Q V I Q L R T R K W T D Q S G Q Y T T E V V N V G T M M L G G R residue Tm 3 Tb 3 Q G A A G I Q G G Q Q Q G G N Q F S G A Q S R Q Q S A A A S E M D F D I F S4

5 Table S3. Δχ tensors determined from the PCSs of Table S2 a Δχ ax /10 32 m 3 Δχ rh /10 32 m 3 Tm 3+ Tb a The Δχ tensors were determined from the PCSs of Table S2 by fitting to the coordinates of the chain A of SSB in the crystal structure 1EYG (9). The PCSs were measured at ph 3.4 and 25 C for 15 N-labeled SSB E65C/E69D ligated with either a C1-Tm or a C1-Tb tag, using a sample with C1-Y tag as the diamagnetic reference. The fits were performed using the program Numbat (32), restricting the lanthanide position to be the same for the Tb 3+ and Tm 3+ data sets. S5

6 13 C ) A S R G V N K V I L V G N L G Q D P E V R Y M P N G G A V A N I T L A T S E S W R D K A T G E M K 13 C ) 13 1 H ) CSI SS E Q T E W H R V V L F G K L A E V A S E Y L R K G S Q V Y 13 C ) I E G Q L R T R K W T D Q S G Q D R Y T T 13 C ) 13 1 H ) CSI SS E V V V N V G G T M Q M L G G R Q G G G A P A G G N 13 C ) I G G G Q P Q G G W G Q P Q Q P Q G G N Q F S G 13 C ) 13 1 H ) CSI SS G A Q S R P Q Q S A P A A P S N E P P M D F D D D I 13 C ) P F 13 C ) 13 1 H ) CSI SS Figure S1. Secondary structure analysis of SSB. The figure was prepared using the program CCPNMR Analysis (30). The rows labeled δ( 13 C α ), δ( 13 C β ), δ( 13 C ), and δ( 1 H α ) indicate the chemical shift differences observed for any given residue with respect to the random coil S6

7 values of C α, C β, C, and H α resonances, respectively. The row labeled CSI reports the chemical shift indices (38). The segments of regular secondary structure in the chain A of the crystal structure 1EYG (9) are depicted underneath. Figure S2. Calculated versus experimental PCSs for the PCS-Rosetta structure that best fulfils the NMR data. The PCS data from residues in the OB-domain are shown in black while PCSs from residues in the C-peptide are shown in red. The left and right panels show the data for Tb 3+ and Tm 3+, respectively. S7

8 Figure S3. Revision of the 2.2 Å X-ray crystal structure of presumably proteolysed full length SSB (12). (A) Original structure, PDB code 1QVC; R = 0.247, Rfree = The OBfold is shown in cyan, except that regions where the amino acid sequence is out of register with the electron density (from residue 90) are shown in red. The orange segments of the Cdomain show weak and discontinuous electron density, with few clear protein protein or protein solvent interactions. (B) Revised structure, based on the deposited structure factors (PDB code 4MZ9), showing only the OB-fold (residues 1 114); R = 0.210, Rfree = S8

9 Supplementary References: 9. Raghunathan,S., Kozlov,A.G., Lohman,T.M. and Waksman,G. (2000) Structure of the DNA binding domain of E. coli SSB bound to ssdna. Nat. Struct. Biol., 7, Matsumoto,T., Morimoto,Y., Shibata,N., Kinebuchi,T., Shimamoto,N., Tsukihara,T. and Yasuoka,N. (2000) Roles of functional loops and C-terminal segments of a single-stranded DNA binding protein elucidated by X-ray structural analysis. J. Biochem., 127, Vranken,W.F., Boucher,W., Stevens,T.J., Fogh,R.H., Pajon,A., Llinas,M., Ulrich,E.L., Markley,J.L., Ionides,J. and Laue,E.D. (2005) The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins, 59, Schmitz,C., Stanton-Cook,M.J., Su,X.C., Otting, G. and Huber, T. (2008) Numbat: an interactive software tool for fitting Dc-tensors to molecular coordinates using pseudocontact shifts. J. Biomol. NMR, 41, Wishart,D.S., Sykes,B.D. and Richards,F.M. (1992) The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy. Biochemistry, 31, S9