Supporting Protocol This protocol describes the construction and the force-field parameters of the non-standard residue for the Ag + -site using CNS

Transcription

1 Supporting Protocol This protocol describes the construction and the force-field parameters of the non-standard residue for the Ag + -site using CNS CNS input file generatemetal.inp: remarks file generate/generatemetal.inp remarks Sample: generate protein structure with metal cluster {*Append the metal cluster*} autogenerate angles=true end {*topology. *} MASS AG ! silver 1+ {*from cns/libraries/toppar/ion.top*} RESIdue AG1 {silver 1+} GROUp ATOM AG+1 TYPE=AG+1 CHARge=+1.0 END END {AG1} {*from cns/libraries/toppar/ion.top*} {*Generate the patch topology,*} {*which will be used in the *} {*PATCh command. *} END end PREsidue AGSM parameter ADD BOND 4AG+1 1SD ADD BOND 4AG+1 2SD ADD BOND 4AG+1 3SD ADD ANGLe 1CG 1SD 4AG+1 ADD ANGLe 2CG 2SD 4AG+1 ADD ANGLe 3CG 3SD 4AG+1 ADD ANGLe 1CE 1SD 4AG+1 ADD ANGLe 2CE 2SD 4AG+1 ADD ANGLe 3CE 3SD 4AG+1 ADD ANGle 1SD 4AG+1 2SD ADD ANGle 1SD 4AG+1 3SD ADD ANGle 2SD 4AG+1 {*Append parameters for metal site*}! eps sigma eps(1:4) sigma(1:4)! (kcal/mol) (A)! NONBonded AG ! Beate, {*from cns/libraries/toppar/ion.param*} nbonds {*This statement specifies the*} atom cdie shift eps=1.0 e14fac=0.4 {*nonbonded interaction energy*} cutnb=7.5 ctonnb=6.0 ctofnb=6.5 {*options. Note the reduced *} nbxmod=5 vswitch {*nonbonding cutoff to save *} end {*CPU time. *} end {*Split the coordinate file into*}

2 {*two files, one containing the *} {*protein coordinates, the other*} {*the metal cluster *} {*coordinates. *} {*First, generate protein.*} segment name="silb_nm2" end end segment name="ag" chain end end the metal*} {*Now generate metal cluster.*} {*Now generate the *} {*covalent links between the protein and patch AGSM reference=1=( resid 34 ) reference=2=( resid 49 ) reference=3=( resid 51 ) reference=4=( resid 200 ) end write coordinates output=ag_silbnm2.pdb end write structure output=ag_silbnm2.psf end stop Associated forcefield parameters: BOND AG+1 SM {sd= 0.001} 2.53 ANGle SM AG+1 SM {sd= 0.031} !(2qcp and 3nsd as examples) ANGle CH2E SM AG {sd= 0.031} !(2qcp and 3nsd as examples) ANGle CH3E SM AG {sd= 0.031} !(2qcp and 3nsd as examples) NONBonded AG !{*from cns/libraries/toppar/ion.param*}

3 Table S1: NMR structural statistics for Ag + -SilB-NM2 (20 structures) I. Experimental constraints Distance restraints Unambiguous distance restraints total number 4065 unique atom pairs 2375 Intraresidual 1708 Sequential 1188 medium range 332 long range 837 Ambiguous distance restraints 419 Dihedral angle restraints Phi 87 Psi 87 II. Constraint violations Distances Rms Å largest violation 3.9 Å Dihedral angles Rms largest violation III. Geometry Mean deviation from ideal geometry bond lengths 5.21 E-3 Å bond angles 0.69 Impropers 1.95 Disallowed 0.3 %

4 Figure S1

5 Figure S1 (continued) Figure S1. Multiple-sequence alignment of CusB (P77239) and CusF (P77214) from E. coli, ZneB (Q1LCD7) and SilB (Q58AF3) from C. metallidurans CH34, and the protein constructs used in this study. Based on the sequence alignment with CusB from E. coli, some functional domains were located on the SilB sequence and represented in different colors: light magenta, N-terminal domain; light red, membrane proximal domain; light blue, C-terminal CusF-like domain. The amino acid residues involved in metal ion binding in CusB, CusF, and ZneB are boxed in red. Regions around the N- and C-terminal metal binding sites in SilB are boxed in black. The additional residues forming the GS loop are boxed in green. The additional residues associated to the human rhinovirus 3C protease cleavage site are shaded in light green.

6 Figure S2 I. II. Figure S2. I. Separation of the purified recombinant SilB and SilB protein constructs by SDS-PAGE. Lane 1, molecular mass standards; lane 2, purified recombinant SilB (A), SilB-NMC (B), SilB-NM (C) and SilB-NM2 (D). The proteins were detected by Coomassie blue (A, B, and D) or silver (C) staining. II. Determination by mass spectrometry of the molecular masses of the purified recombinant SilB (A), SilB-NMC (B), SilB-NM (C) and SilB-NM2 (D). The insets represent the molecular mass spectra obtained after deconvolution of the raw data using the MaxEnt1 software (Waters). The experimental masses are in good agreement with the theoretical masses calculated for the recombinant proteins devoid of the N-terminal methionine residue: SilB ( Da), SilB- NMC ( Da), SilB-NM ( Da), and SilB-NM2 ( Da).

7 Figure S3 Figure S3. ESI mass spectra of the recombinant proteins in 10 mm ammonium acetate (ph 6.9) titrated with metal ions. The protein concentration was 5 µm. (A) [M + 16H] 16+ charge state of SilB in the apo form (1) and in the presence of 1 molar equiv (2) and 2 molar equiv (3) of Ag +, and 1 molar equiv (4) and 2 molar equiv (5) of Cu +. (B) [M + 11H] 11+ charge state of SilB-NMC in the apo form (1) and in the presence of 2 molar equiv of Ag + (2) and Cu + (3). (C) [M + 9H] 9+ charge state of SilB-NM in the apo form (1) and in the presence of 1 molar equiv of Ag + (2) and Cu + (3). (D) [M + 8H] 8+ charge state of SilB-NM2 in the apo form (1) and in the presence of 1 molar equiv of Ag + (2) and Cu + (3). The position of the apo form peak is depicted by a dashed line and the red arrows represent the mass shifts observed upon the binding of metal ions.

8 Figure S4 Figure S4. Isothermal titration calorimetry: Titration of AgNO 3 into SilB-NM2 (A) in MilliQ water at 25 ºC, and SilB-C (B) in 20 mm HEPES ph 7.0 at 25 ºC. Top panel, differential heating power versus time. Lower panel, integrated and normalized heat of reaction versus the molar ratio. Experimental data are represented by black squares ( ). Lines show the best fit to the binding isotherm using a single-site binding model.

9 Figure S5 Figure S5. 1 H, 13 C-HSQC spectra centred on the region in which methionine methyl correlation peaks show up. (A) Titration of SilB-NM (black: apo, green: molar equiv, cyan: molar equiv, and red: molar equiv of Ag + ions). (B) 1 H, 13 C-HSQC spectra of apo-silb-nm2 (black) and Ag + -SilB-NM2 (violet). Arrows indicate shifts that occur upon metal binding, indicating that the three labeled methionine residues are involved in binding of the Ag + ion (sequence numbering of SilB-NM2).

10 Figure S6 Figure S6. Superposition of 1 H, 15 N-HSQC spectra of SilB-NMC (blue), SilB-NM (green) and SilB- C (red) in the absence (top) and the presence of 1 molar equiv of Ag + ions per binding site (bottom). Nearly all cross-peaks of the SilB-NMC spectrum have an equivalent in one of the two other spectra. The boxed region contains side chain resonances that are refolded differently as a function of the 15 N spectral width.

11 Figure S7 Figure S7. Ag + -titration of SilB-C. Superposition of 1 H, 15 N-HSQC spectra acquired with 0 (blue), 0.5 (green) and 1 (magenta) molar equiv of Ag + ions added. Cross-peaks are labeled according to the previous NMR study of the CusF-like domain. 30

12 Figure S8 Figure S8. Weighted chemical shift differences between amide groups of the C-terminal domain of apo-silb-nmc and SilB-NMC in the presence of 1 molar equiv of AgNO 3 (half-saturated SilB- NMC) as a function of SilB sequence.

13 Figure S9 Figure S9. ESI mass spectra of a mixture of 5 µm SilB-C and 5 µm SilB-NM in 10 mm ammonium acetate (ph 6.9). (A) Multiple-charge ion mass spectrum where the peaks corresponding to apo SilB-C and SilB-NM are labeled with open squares and stars, respectively. (B) Zoom of this spectrum in regions where the [M + 6H] 6+ charge state of SilB-C and the [M + 9H] 9+ charge state of SilB-NM are detected (1). Upon addition of 5 µm of Ag +, the metal ions are mainly associated to SilB-C (2), while both proteins are fully metallated in the presence of 10 µm of Ag + (3). The metalbound forms of both proteins are represented with filled symbols.

14 Figure S10 Figure S10. Domain-domain interactions monitored by isothermal titration calorimetry. (A) Titration of apo-silb-c (100 μm) into Ag + -SilB-NM (10 μm). (B) Titration of apo-silb-nm (100 μm) into Ag + -SilB-C (10 μm). (C) Titration of apo-silb-c (100 μm) into apo-silb-nm (10 μm). (D) Titration of Ag + -SilB-C (100 μm) into Ag + -SilB-NM (10 μm). Top panel, differential heating power versus time. Lower panel, integrated and normalized heat of reaction ( ) versus the molar ratio of the two proteins.

15 Figure S11 Figure S11. (A) Metal ion transfer between Ag + -SilB-NM and apo-silb-c monitored by native mass spectrometry. The mass spectra represent the regions corresponding to the [M + 6H] 6+ charge state of SilB-C and the [M + 9H] 9+ charge state of SilB-NM as described in Figure S9. Peaks associated to SilB-C and SilB-NM are represented with open squares and stars, respectively. The metal-bound forms of both proteins are represented with filled symbols. (1) 5 µm of Ag + -SilB-NM in 10 mm ammonium acetate, ph 6.9. (2) Upon addition of 5 µm of apo-silb-c to this latter, a large amount of Ag + bound to SilB-NM is transferred to SilB-C. The transfer of metal ions between the two proteins is significantly less efficient when 5 µm of Ag + -SilB-C (3) are incubated in the presence of 5 µm apo-silb-nm (4). (B) Identical experiments realized by replacing SilB-NM with SilB-NM2. The mass spectra represent the regions corresponding to the [M + 6H] 6+ charge state of SilB-C and the [M + 8H] 8+ charge state of SilB-NM2. Peaks associated to SilB-C and SilB-NM2 are represented with open squares and circles, respectively. The metal-bound forms of both proteins are represented with filled symbols.

16 Figure S12

17 Figure S12 (continued) Figure S12. Metal exchange between Ag + -SilB-NM and apo-silb-c. (A) 1 H, 15 N-HSQC spectrum of 0.18 mm 15 N-labeled SilB-NM to which 0.1 mm AgNO 3 (compare S10B) and 0.11 mm 13 C, 15 N- labeled apo-silb-c had been added consecutively. Specific resonances of apo-silb-nm and Ag + - SilB-C are indicated by black and green arrows, respectively. (B) 1 H, 15 N-HSQC spectra of 0.18 mm SilB-NM in absence (black) and presence of 0.1 mm AgNO 3 (red). (C) Control spectrum of apo-silb-c (green) compared to Ag + -SilB-C (violet). (D) 1 H, 13 C-HSQC spectra of 13 C, 15 N-labeled apo-silb-c (green) and the mixture of 0.18 mm 15 N-labeled SilB-NM, 0.1 mm AgNO 3 and 0.11 mm 13 C, 15 N-labeled apo-silb-c (blue). Arrows indicate positions of the methyl groups of the two methionines involved in the Ag + -site. The blue spectrum shows the characteristic downfield shift indicating that Ag + ions are bound by the methionines. The spectra shown in A, B and D have been obtained with the same SilB-NM sample during a titration with AgNO 3 and apo-silb-c.

18 Figure S13 Figure S13. Quantification of slow chemical exchange rates for Ag + -SilB-NM2. (A) Extract of the two-dimensional EXSY spectra acquired with mixing times of 0 (dark grey), 50 ms (green) and 250 ms (red). (B) Result of the data analysis in which peak intensities measured as a function of mixing time were fitted to a two-site exchange model. Experimentally measured longitudinal relaxation rates were considered in the fitting process. Measured peak intensities are shown as a function of mixing time for the diagonal peaks AA (red), BB (violet) and the exchange peaks AB (blue) and BA (black). The results of the best fit obtained with p A = 0.53±0.05, p B = 0.47±0.05, k AB = 7.5±1.0 s -1, k BA = 8.4±1.1 s -1 are shown as continuous lines.

19 Figure S14 Figure S14. Absolute chemical shift differences between backbone atoms of Ag + -bound SilB-NM2 form A and form B. Carbon-α (blue triangles), carbonyl carbon (black squares), and amide nitrogen (green dots).

20 Figure S15 Figure S15. The structure and environment of the membrane proximal domain. (A) Comparison of the three-dimensional structures of the MP domain from SilB (red), E. coli CusB (cyan) (PDB code 3NE5) and C. metallidurans ZneB (violet) (PDB code 3LNN). N and C-termini of the individual domains are indicated in the corresponding colors. For CusB and ZneB, the neighboring -barrel domain is indicated. (B) The MP domain from CusB in the supramolecular CusAB 2 complex (PDB code 3NE5). The molecular surface of CusA is shown in blue, those of the two CusB chains from the same asymmetric unit in pink. The surface of the MP domains is colored according to the electrostatic potential ranging from -3 kt (red) to +3 kt (blue). The N- and C-termini of each MP domain are indicated. The approximate location of the membrane is indicated in green. (C) The CusA 3 B 6 complex in a different orientation.