Metal-dependent allosteric activation and inhibition on the same molecular scaffold: the copper sensor CopY from Streptococcus pneumoniae

The dynamics and marginal stability of CopY enable allosteric activation of DNA binding by Zn(ii) and inhibition by Cu(i).


SUPPLEMENTAL METHODS
CopY expression and purification. A pHis plasmid was used to subclone wild-type, C101A and C52A/C101A CopY from S. pneumoniae D39 strain (locus tag SPD_0633). This vector encodes a His 6 tag and TEV protease cleavage site at the N terminus to yield, following cleavage, three non-native N-terminal amino acids appended to the sequence GAM for CopY. 1 The same strategy was used to subclone the C-terminal regulatory domain comprising residues 68-131. BL21(DE3) competent cells were transformed with the indicated plasmids. For unlabeled proteins, an overnight culture of E. coli was inoculated into Luria Broth (LB) media containing 100 µg/mL ampicillin. For uniformly 15 N-labeled or 13 C, 15  The cells were harvested by low speed centrifugation and kept at -80 °C until use. All the buffers in the purification were degassed and refilled with Argon using a Schlenk line just before use. To break the cells, they were resuspended in buffer R (25 mM Tris, pH 8.0, 200 mM NaCl, 5 mM TCEP) and lysed by a sonic dismembrator (Fisher). The recombinant proteins were purified using Ni-charged HisTrap FF columns (GE Healthcare) using a gradient of imidazole from 25 mM to 325 mM in buffer R. The appropriate fractions were pooled and subjected to TEV protease cleavage at for 16 °C for 36 h and reapplied to the HisTrap FF column, with the flow-through fractions collected and chromatographed on a Superdex 75 16/60 column (GE Healthcare). The purity of the proteins was estimated to be ≥90% as judged by SDS-PAGE and ESI-MS (Fig. S1A), reduced to a level of ≥90% using Ellman's reagent (DTNB) to detect free thiols 2 and metal-free by atomic absorption spectroscopy or ICP-MS. All CopY preparations contained a trace of what appears to be irreversibly cross-linked dimer on an SDS-PAGE gel, likely the origin of less than fully quantitative recovery of free thiols (Fig. S1A). Protein concentration was determined by A 280 with an extinction coefficient of 18,240 M -1 cm -1 on a UVvisible spectrometer (HP/Agilent 8453 spectrophotometer). µL aliquot of titration solution was mixed with increasing CopY titrant. Optical spectra of BCS were recorded from 200 nm to 900 nm. Corrected spectra were obtained by subtracting baseline spectrum from each CopY addition spectrum, and then corrected for dilution. A483 was used to determine the concentration of Cu2-BCS complex with an extinction coefficient of 13,500 M -1 cm -1 . 3 All the data were fitted to the appropriate competition model using Dynafit 4 as previously described.

Zn(II) binding affinity measurements.
These experiments were carried out as described previously using absorption spectroscopy (A 325 for apo-mf2; A 366 for Zn(II)-mf2) of mag-fura-2 (mf2). 5,6 Briefly, anaerobic titrations of a ZnSO 4 stock into a solution of a known concentration of apo-C101A CopY and mf2 were carried out and the resultant data fit to a 1:1 (Zn:CopY dimer) chelator competition model using Dynafit 4 , assuming a non-dissociable CopY dimer.
X-ray absorption spectroscopy. C101A CopY was used for all of these experiments.
Apo-CopY samples were incubated with 0.5 protomer mol equivalents (1 per dimer) Zn(II) (Zn 1 CopY), or 1 protomer mol equivalents (2 per dimer) Cu(I) prepared in NaBr (Cu 2 CopY-Br) or NaCl (Cu 2 CopY-Cl) were concentrated to 800 µM, 1. Cu 2 CopY-Cl) were loaded into an aluminum sample holder, which was cooled to ~50 K by using a He displex cryostat. Data were collected under ring conditions of 2.8 GeV and 120-300 mA using a sagitally focusing Si(111) double-crystal monochromator. Harmonic rejection was accomplished with a Ni-coated focusing mirror. X-ray fluorescence was collected using a 30element Ge detector (Canberra). Scattering was minimized by placing a Z-1 filter between the sample chamber and the detector. Data at the SSRL (Cu 2 CopY-Br) were collected at 10 K using a liquid helium cryostat (Oxford Instruments) with ring conditions of 3 GeV and 80-100 mA. Beamline optics consisted of a Si(220) double-crystal monochromator and two rhodiumcoated mirrors, a flat mirror before the monochromator for harmonic rejection and vertical columnation, a second toroidal mirror after the monochromator for focusing. X-ray fluorescence was collected using a 30-element Ge detector (Canberra). Scattering was minimized by using Soller slits and placing a Z-1 filter between the sample chamber and the detector. XANES were collected from ± 200 eV relative to the metal edge. The X-ray energy for each metal K α -edge was internally calibrated to the first inflection point of the corresponding metal foil for Cu, 8980.3 eV, and Zn, 9660.7 eV. EXAFS was collected to 15 k above the edge energy (E o ).

XAS Data Reduction and Analysis.
The XAS data shown (vide infra) are the average of 5 and 7 scans for Zn 1 CopY and Cu 2 CopY, respectively. XAS data were analyzed using SixPack. 7 The SixPack fitting software builds on the ifeffit engine. 8,9 Each data set was background-corrected and normalized. The EXAFS equation is defined as (eq 1): where f(k) is the scattering amplitude, δ(k) is the phase-shift, N is the number of neighboring atoms, r is the distance to the neighboring atoms, and σ 2 is the disorder to the nearest neighbor.
For EXAFS analysis, each data set was converted to k-space using the relationship (eq 2): ( where is the mass of the electron, is Plank's constant divided by 2π, and is the threshold energy of the absorption edge. The threshold energy chosen for copper is 8990 eV and zinc is 9670 eV. 10 The best fits for the data sets were obtained using a Fourier-transform over the range k = 2-12.5 Å -1 , where the upper limit was determined by the signal:noise ratio. Scattering parameters were generated using FEFF 8. 9 The first coordination sphere was determined by setting the number of scattering atoms in each shell to integer values and systematically varying the combination of S, Cl, Br-donors (Tables S1-S3). To compare different models of the same data set, ifeffit utilizes three goodness of fit parameters: χ 2 , reduced χ 2 , and e m ! o E the R-factor. χ 2 is given by eq 3, where is the number of independent data points, N e2 is the number of uncertainties to minimize, is the real part of the EXAFS function, and is the imaginary part of the EXAFS fitting function (eq 3). ( Reduced where N varys is the number of refining parameters and represents the degrees of freedom in the fit. Additionally, Ifeffit calculates the R-factor for the fit, which is given by eq 4, and is scaled to the magnitude of the data making it proportional to χ 2 (eq 4).
In comparing different models, the R-factor and reduced χ 2 parameter were used to determine which model was the best fit of the data. The R-factor will always generally improve with an increasing number of adjustable parameters, while reduced χ 2 will go through a minimum and then increase, indicating that the model is over-fitting the data. 11

Ratiometric Pulsed-Alkylation Mass Spectrometry (rPA-MS). Alkylation step.
Sample preparation was adapted from an earlier report 12  exactly as described in previous work. 13 All EMSA and anisotropy-based data were fit to a simple 1:1, non-dissociable dimer binding model to estimate K a using DynaFit. 4

Bacterial strain construction, growth conditions for S. pneumoniae. The strains in
the study were derived from Streptococcus pneumoniae D39 (IU1781) and are listed in Table S4.
All S. pneumoniae D39 mutants were constructed by gene deletion replacement and counter antibiotic selection using the rpsL+ cassette, Janus 14 as previously described using standard techniques. 15 Brain-heart infusion (BHI) medium was of standard composition and prepared with double distilled water. For growth experiments, bacteria were inoculated into BHI broth from frozen culture stocks, then serially diluted and propagated overnight. The next day, exponentially growing cultures were diluted to approximately 0.005 OD 620 into pre-warmed BHI containing increasing concentrations of CuSO 4 . All aerobic cell growth experiments were monitored over time at 37 °C in an atmosphere of 5% CO 2 .

RNA isolation and quantitative real-time PCR (qRT-PCR).
Total RNA was isolated from cells by hot phenol extraction, DNase digested and converted to cDNA as previously described. 6 PCR amplification was carried out as previously described using the copA forward and reverse primers. gyrA served as the housekeeping gene. 16 PCR outcomes were normalized to the gyrA gene and relative transcript levels were calculated by comparison of the ratio of stressed to non-stressed cells.
NMR spectroscopy. Typical NMR sample solution conditions were 300-600 µM 15 N-or 15 N/ 13 C-labeled wild-type, C101A or C52A/C101A CopY in 20 mM HEPES, 0.2 M NaCl, 5 mM TCEP, pH 6.0 as indicated. All NMR samples were prepared in an anaerobic glove box. Apo-CopY was prepared with 5 mM TCEP. Agilent 800 and 600 MHz spectrometers equipped with cryogenic probes in the METACyt Biomolecular NMR Laboratory were used to acquire data for all CopY samples. NMR data were processed using NMRPipe and were analyzed using Sparky 17 . All spectra were acquired at 25 °C or 30 ºC as indicated. Chemical shift is referenced relative to 2,2-dimethyl-2-silapentene-5-sulfonic acid (DSS).

Small angle x-ray scattering (SAXS). The apo, Zn 1 and Cu 2 binding states of C101A
CopY were prepared using the same protocol as described in the rPA-MS experiments. All Hampton, MA). The IM spectra shown in Fig. 9 were background subtracted integrating the signal before each peak and fitted to Gaussian peaks. Table S1. Selected EXAFS fits for Cu 2 CopY dimer in buffer containing NaCl. Data fit from k = 2-12.5 Å -1 and r = 1-4 Å.