Analyses of Mlc–IIB Glc interaction and a plausible molecular mechanism of Mlc inactivation by membrane sequestration

Nam et al. 10.1073/pnas.0709295105.

Supporting Information

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SI Table 1
SI Figure 5
SI Figure 6
SI Figure 7
SI Figure 8
SI Figure 9
SI Materials and Methods




SI Figure 5

Fig. 5. Close-up view of the clasping tetrameric interface around Arg-306 and Leu-310. For clarity, an Mlc dimer is covered by a transparent surface.

SI Figure 6

Fig. 6. Determination of oligomeric state of Mlc. (A) Oligomeric state of Mlc and an Mlc mutant was analyzed by gel filtration chromatography. The proteins were eluted with 10 mM Tris buffer (pH 7.5) containing 1 mM DTT and 100 mM NaCl. The flow rate was 1.5 ml/min. wtMlc and mMlc represent wild-type and mutant Mlc, respectively. An enormous peak at about 32 min contains no proteins according to SDS/PAGE. The enormous peak may represent nonprotein aggregates that are generated during the refolding process of wtMlc. (B) The line fitting of the elution time versus logarithm of the molecular weight of the size markers. Predicted molecular mass of an Mlc mutant is 93,325 Da, indicating that the mutant exists as a dimer in solution.

SI Figure 7

Fig. 7. Stereo view of the superimposed Ca tracing of the HTH motif-containing parts in BlaI (red) and Mlc (green). The rms deviation of Ca atoms is 1.2 Å. The secondary structure labels are for Mlc. a2 and a3 form a HTH motif. The structural homologue search of the Mlc D-domain using the program DALI identified 358 homologous DNA-binding domains exhibiting z-scores of >2. Among top-ranked 20 structures (z-scores >7), we tried to find out a protein-DNA complex structure where two recognition helices of a protein fit into tandem major grooves of DNA. The BlaI/DNA complex structure was selected because this is the only one satisfying such a criterion.

SI Figure 8

Fig. 8. Phosphorylation state-dependent interaction between Mlc proteins and the GrpE-EIIB fusion protein. GrpE-EIIB was immobilized onto the carboxymethylated dextran surface of a CM5 sensor chip to a surface concentration of 3 ng/mm². The standard running buffer was 10 mM Hepes (pH 7.2), 150 mM NaCl, 10 mM KCl, 2 mM DTT, 1 mM MgCl₂, and 0.5 mM EDTA, and all reagents were introduced at a flow rate of 10 ml/min. The sensor surface was regenerated between assays by flowing 2 M NaCl at a flow rate of 100 ml/min for 10 min to remove bound analytes. To phosphorylate the immobilized GrpE-EIIB, a mixture (50 ml) of phosphoenolpyruvate (PEP) (0.5 mM), EI (20 mg/ml), HPr (10 mg/ml), and EIIA^Glc (10 mg/ml) in standard running buffer was allowed to flow into the flow cell for 10 min. The O-domain mutant of Mlc (Arg306Gly and Leu310Gly, 100 mg/ml) was allowed to flow over the GrpE-EIIB surface for 8 min in each sensorgram. The phosphorylated and dephosphorylated GrpE-EIIB surfaces were generated by reversible phosphoryl transfer reactions between EIIA^Glc and EIIB as described above. The sensorgram in Left shows the mutant Mlc binding to the immobilized GrpE-EIIB surface without any treatment. In the sensorgram in Right, the mutant Mlc was injected after the immobilized GrpE-EIIB surface had been phosphorylated by exposing it to a mixture of EI, HPr, and EIIA^Glc in the presence of phosphoenolpyruvate (PEP), then flushing with running buffer to remove PEP and other PTS proteins. It should be noted that wild-type Mlc also showed the phosphorylation state-dependent interaction with the GrpE-EIIB fusion protein (data not shown).

SI Figure 9

Fig. 9. Electrophoretic mobility shift assay showing that the O-domain Mlc mutant efficiently binds to the Mlc target site. ³²P-labeled DNA fragments (17 nM) covering the Mlc binding region of the ptsG promoter (from -260 to -155 with respect to the transcriptional start site) were mixed with wild-type and the O-domain mutant of Mlc and resolved in a 6% polyacrylamide gel as indicated. The concentration of the Mlc proteins is at 0.25 mM (lane 2), 0.5 mM (lane 3), 1.0 mM (lane 4), and 2.0 mM (lane 5). The arrowhead indicates the DNA associated with the Mlc proteins.

Table 1. Data collection and refinement

 

¹The number in parentheses is for the outer shell

²R_sym = ∑_∑h∑_∑I |I_h,i - I_h|/∑_∑h∑_∑I∑∑I_h,i, where I_h is the mean intensity of the i observations of symmetry related reflections of h.

³R = ∑ |F_o-F_c|/∑ F_o, where F_o = F_p, and F_c is the calculated protein structure factor from the atomic model. R_free was calculated with 10% of the reflections.

⁴ rms deviations in bond length and angles are the deviations from ideal values.

SI Materials and Methods

Cloning. For the production of selenomethionine (SeMet)-labeled EIIB, the DNA fragment encoding EIIB was removed from the pJHK vector (1) and cloned into the pET-22b(+) vector (Novagen) at the NdeI and BamHI sites. Protein was expressed in Escherichia coli B834(DE3)Dcrr constructed by P1 transduction of the crr (Km^R) locus from TSDH02 (2) into B834 for selenomethionine (SeMet) incorporation. The pRE1-GST plasmid used for the expression of E. coli GST was constructed by cloning gst into pRE1 vector (3) as NdeI/BamHI fragment from E. coli genomic DNA amplified by PCR using a mutagenic forward primer to create an NdeI site in front of the start codon and a mutagenic primer to create a BamHI site 11 nt downstream of the TAA stop codon. GST-EIIB was expressed by using a plasmid constructed by the insertion of entire coding region of E. coli GST except last 9 nt including the TAA stop codon into the upstream of EIIB-coding sequence in frame in the NdeI site of pJHK plasmid (1). Similarly, Grx2-EIIB and GrpE-EIIB were expressed by using plasmids constructed by the insertion of entire coding regions of E. coli Grx2 and GrpE except the stop codons, respectively, into the upstream of EIIB coding sequence in frame in the NdeI site of pJHK plasmid. Site-directed mutagenesis was used to create point mutations in EIIB and Mlc.

Crystallization. Crystals of EIIB were crystallized at 23°C with mother liquors of 3.2 M ammonium sulfate and 0.1 M citric acid (pH 4). The crystals belonged to the space group C222₁ with cell parameters a = 108.1, b = 108.1, and c = 28.9 Å. Crystals of the Mlc/EIIB complex were obtained at 23°C with mother liquors of 6% PEG 6K, 0.1 M MgCl₂, and 0.1 M sodium acetate (pH 5.5). The crystals belonged to the space group C2 with cell parameters a = 201.4, b = 55.4, c = 82.5 Å, and b= 95.3°. For data collection, crystals were frozen at -170°C after a brief immersion in a cryoprotectant solution containing 20% glycerol in the same mother liquor. A 2.85-Å dataset of Mlc/EIIB and a 1.65-Å native dataset of EIIB (SI Table 1) were collected by using a Bruker Proteum 300 CCD at beamline 6B at Pohang Light Source, Korea, and a 2.1-Å MAD dataset of EIIB (SI Table 1) was collected at beamline 5A at Photon Factory, Japan.

Structure Determination and Refinement. The model building was performed by using XtalView, and refinement was done with the maximum-likelihood algorithm implemented in CNS (SI Table 1) (4). The structure of Mlc/EIIB was determined by molecular replacement using the program MolRep. O-, E-, and D-domains from the Mlc mutant structure and our high-resolution structure of EIIB were used as search models. That is, four independent models were used to obtain phasing information. The model building of the complex was straightforward because there was no sequence difference. The initial rigid-body refinement decreased the R value to 29.5%. Several rounds of refinement (which included a weak harmonic restraint for all protein atoms, harmonic restraints constant = 10) and remodeling produced a final model refined to R and R_free values of 22.8% and 30.1%, respectively.

The four selenium positions of crystals of SeMet-labeled EIIB were located, and phases were calculated by using the program Solve. The subsequent solvent flattening by Resolve gave rise to an interpretable map, based on which the de novo model building of EIIB was easily completed. The initial model was subjected to a rigid body and a positional refinement. The R value dropped to 27.2%. At this stage, the peak data were replaced by 1.65-Å native data and water molecules and a sulfate ion were incorporated into the corresponding density. The subsequent refinement and manual refitting of model reduced R and R_free values to 21.3% and 24.1%, respectively.

Surface Plasmon Resonance Spectroscopy. Surface plasmon resonance experiments were performed on a BIAcore 3000 instrument. Wild-type and mutant EIIB proteins were separately immobilized on a CM5 sensor chip as described (1). Response differences during immobilization were 760 RUs (wild type), 400 RUs (V449Q), 930 RUs (A451F), and 950 RUs (Q456A). Running buffer was 10 mM Hepes (pH 8.0) containing 200 mM NaCl, 10 mM KCl, 1 mM MgCl₂, 1 mM DTT, and 0.005% Tween 20, and all reagents were introduced at a flow rate of 10 ml/min. The sensor surface was regenerated between injections by flowing the running buffer containing 100 mg/ml wild-type EIIB or 100 mg/ml A451F mutant of EIIB for 4.5 min. Kinetic parameters were determined by fitting to a 1:1 Langmuir binding model using BIAevaluation 3.1 software (Biacore) assuming that the Mlc tetramer binds to the immobilized EIIB.

1. Nam T-W, et al. (2001) The Escherichia coli glucose transporter enzyme IICB^Glc recruits the global repressor Mlc. EMBO J 20:491-498.

2. Nam T-W, Park Y-H, Jeong H-J, Ryu S, Seok Y-J (2005) Glucose repression of the Escherichia coli sdhCDAB operon, revisited: Regulation by the CRP-cAMP complex. Nucleic Acids Res 33:6712-6722.

3. Reddy P, Peterkofsky A, McKenney K (1989) Hyperexpression and purification of Escherichia coli adenylate cyclase using a vector designed for expression of lethal gene products. Nucleic Acids Res 17:10473-10488.

4. Brünger AT, et al. (1998) Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D 54:905-921.

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