File S1 - Post-Transcriptional Regulator Hfq Binds Catalase HPII: Crystal Structure of the Complex

Fig. S1, Crystal of the Hfq and catalase HPII complex and catalase activity of crystals. (A) A typical crystal for X-ray diffraction experiments. Bar represents 100 µm. (B) Assay curve for crystals of the Hfq and catalase HPII complex. Decomposition of hydrogen peroxide (H2O2) over time shows the decrease in absorbance at λ = 240 nm. Time = 0 indicates a point just after H2O2 was added. Fig. S2, Analytical gel-filtration of Hfq and HPII. (A) Dashed line: wild-type Hfq after incubation at 40°C. Thin line: mixture of wild-type Hfq (∼0.5 mg/ml) and HPII (∼0.5 mg/ml) at 4°C. Thick line: the same sample as shown in the thin line, but after incubation at 40°C. The peak in the dashed line corresponds to 138 kDa and the earlier eluting peak at ∼1.7 ml in thin line corresponds to ∼320 kDa as calibrated with soluble globular proteins used as standards. (B) SDS-PAGE patterns of overproduced and purified Hfq and HPII. Lanes are: 1, marker; 2, Hfq; 3, HPII; and 4, the peak fraction of analytical gel-filtration of mixture of Hfq and HPII shown in the thick line in (A). Arrows are: upper, HPII; middle, hexamer of Hfq; and lower, monomer of Hfq. Other bands probably indicate degradation of HPII after incubation at 40°C. Fig. S3, Typical electron micrographs of protein complexes prepared with negative staining of the peak fraction in thick line in Fig. S2. Purified Hfq and HPII were mixed and incubated at 40°C and gel-filtrated. Protein complexes with ring-like structures are similar to complexes of endogenous Hfq and HPII (Fig. 1B). Bar refers to 100 Å. Fig. S4, Interactions of Hfq subunits 2 and 6 with HPII. (A) Subunit 2 in green. (B) Subunit 6 in green. The figures display the interface between the distal surface of Hfq and the core domain of HPII. The representation scheme of molecules is the same as in Fig. 5. See also Table S2 for more information on the interactions. Fig. S5, Interactions of Hfq subunits 1 and 4 with HPII. (A) Interface between the distal surface of Hfq subunit 1 (cyan) and the C-terminal lobe of HPII. (B) Interface between the proximal surface of Hfq subunit 4 (green) and lower part of the C-terminal lobe of HPII in a neighboring tetramer. The representation scheme is the same as in Fig. 5. Densities for the side chains of Glu 610 are not visible at this contour level. Table S1, Data collection and refinement statistics for the E.coli Hfq-catalase HPII complex. Table S2, Major interactions between Hfq and catalase HPII. SI Materials and methods. (PDF)