SUPPLEMENTARY INFORMATION

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1 Supplementary Figure 1: The HpUreI crystal used for collection of native diffraction data. The crystal belongs to spacegroup P and has an approximate maximal dimension of 0.25 mm. Supplementary Figure 2: Quantitative Western blot analysis using purified recombinant HpUreI as a standard and antibody against HpUreI. Five and ten micrograms of bacterial cell suspension were loaded and run side-by-side with varying concentrations of purified HpUreI. The Western blot was scanned to determine the optical density of the bands and a standard curve of nanograms of purified HpUreI versus integrated optical density was plotted and used for estimation of the cellular content of HpUreI. 1

2 Supplementary Figure 3: Native gel (PAGE) of HpUreI supports the hexameric structure in solution. However, due to unknown amount of bound detergent/lipid, a smaller oligomer cannot be excluded. The computed molecular weight without detergent/lipid is 22.5 kda per protomer and 135 kda for the hexamer. 2

3 Urea chemical structure and reaction catalyzed by urease Supplementary Figure 4: Structure of urea and the reaction catalyzed by cytoplasmic urease. Urea is planar and neutral but polar with a dipole moment of 4.56 Debye. 3

4 Experimental electron density map of the HpUreI hexamer The electron density map of the HpUreI hexamer obtained without model phases from SeMet MAD phases is shown in Supplementary Fig. 5. Supplementary Figure 5: Experimental electron density of HpUreI hexamer. Experimental electron density obtained without model phases from SeMet MAD phases after 3-fold noncrystallographic symmetry and multi-crystal averaging coupled with solvent flattening contoured at 1.5 sigma, viewed from the periplasmic side together with a ribbon diagram of the final model. The C6 hexamer is generated from the three protomers of one asymmetric unit (one green, one red & one blue molecule) by the crystallographic two-fold axis. 4

5 The cytoplasmic surface of HpUreI Supplementary Fig. 6 shows the cytoplasmic surface of HpUreI. Supplementary Figure 6: The cytoplasmic surface of HpUreI. Viewed from the cytoplasm, the hydrophobic character of the cytoplasmic channel vestibules becomes apparent (green arrow). This is the side of HpUreI that cytoplasmic urease dodecamers are thought to interact with for efficient urea hydrolysis. 5

6 Electron density map of transmembrane helix 3 Supplementary Fig. 7 shows the electron density map of transmembrane helix 3 (TMH3). Supplementary Figure 7: Electron density map of TMH3. The sharpened 2F o -F c map (sharpening B of 150 Å 2 and an α of 0.3) 9 is contoured at 1 sigma. 6

7 Electron density map of the constriction region around Trp153 Supplementary Fig. 8 shows the electron density map of the constriction region around Trp153. Supplementary Figure 8: Electron density map of the constriction around Trp153. The sharpened 2F o -F c map (sharpening B of 150 Å 2 and an α of 0.3) 9 is contoured at 1 sigma and shows Trp153 occluding the channel. 7

8 A lipid bilayer plug in the center of the HpUreI hexamer The center of the hexamer is filled with a lipid plug that forms a bilayer. The sharpened electron density omit map is shown in Supplementary Fig. 9. Supplementary Figure 9: Electron density map of the lipid plug at center of HpUreI hexamer. Density in the form of long tubes was visible in the center of the hexamer in the initial MAD-phased maps. This figure shows the sharpened omit map computed after refinement without the lipids (sharpening B of 150 Å 2 and an α of 0.3) 9 and is contoured at 1 sigma. There is electron density for six lipid tails in the periplasmic leaflet and for 18 tails in the cytoplasmic leaflet. The plug is narrower on the periplasmic side (17 Å vs. 28 Å diameter), where six copies of TMH2 converge, than on the cytoplasmic side, where the plug is lined by six copies of TMH2 and TMH3 (Supplementary Fig. 10). 8

9 Supplementary Figure 10: Lipid tails at the center of the HpUreI hexamer based on electron density. Side view with HpUreI molecules in foreground removed for clarity. The periplasmic side is on top (6 short lipid tails) and the cytoplasmic side on the bottom (18 longer lipid tails). Amino acid sequence alignment of HpUreI J99 with other proteins from the AmiS/UreI superfamily Multiple sequence alignment of eleven eubacterial and one archaeal sequence from the AmiS/UreI superfamily. Eleven residues are totally conserved: three from TMH1, four from TMH3, one from TMH4 and three from TMH5 (Supplementary Fig. 11). Multiple sequence alignment of eight sequences from the UreI family that are known to be urea channels (Supplementary Fig. 12). 9

10 Supplementary Figure 11: Multiple sequence alignment of eubacterial and archaeal sequences from the AmiS/UreI superfamily. The secondary structure cartoon above the alignment is based on the H. pylori UreI crystal structure. Residue numbering at the bottom corresponds to the H. pylori J99 UreI sequence. Sequences were aligned with the ClustalW program 1 and shaded with the TeXshade program 2. The top three sequences represent proteins from the UreI family of urea channels. The bottom sequence is from the archaeon Ferroglobus placidus. The highly conserved sequences discussed in the text are designated by identity in at least 11 out of 12 sequences. Organisms and accession numbers of sequences used in the alignment: H. pylori (Helicobacter pylori J99 strain, NP_222788), H. hepaticus (Helicobacter hepaticus, NP_859940), S. salivarius (Streptococcus salivarius, AAC72025), P. aeruginosa (Pseudomonas aeruginosa, NP_ ), B. parapertussis (Bordetella parapertussis, NP_ ), P. denitrificans (Paracoccus denitrificans, YP_ ), D. 10

11 acidovorans (Delftia acidovorans, YP_ ), N. eutropha (Nitrosomonas eutropha, YP_ ), Arthrobacter (Arthrobacter sp FB24, YP_ ), N. farcinica (Nocradia farcinica, YP_ ), B. cereus (Bacillus cereus, NP_ ), F. placidus (Ferroglobus placidus DSM 10642, ADC64572). Color code used for shading: black: fully conserved; yellow: >50% conserved; light blue: >50% similar. Supplementary Figure 12: Multiple sequence alignment of bacterial homologs from the UreI family that are known to be urea channels. The secondary structure cartoon above the alignment is based on the H. pylori UreI crystal structure. Residue numbering at the bottom corresponds to the H. pylori J99 UreI sequence. Sequences were aligned with the program ClustalW 1 and shaded with the program TeXshade 2. Organisms and accession numbers of sequences used in the alignment: H. pylori (Helicobacter pylori J99 strain, NP ), H. pylori (Helicobacter pylori strain, NP ), H. bizzozeronii (Helicobacter bizzozeronii, AAO15375), H. felis (Helicobacter felis, ABI95467), H. hepaticus (Helicobacter hepaticus,np ), H. bilis (Helicobacter bilis, ZP ), H. mustelae (Helicobacter mustelae, YP ), S. salivarius (Streptococcus salivarius, AAC72025). Color code used for shading: black: fully conserved; yellow: >50% conserved; light blue: >50% similar. 11

12 Electrostatic potential on the periplasmic surface Electrostatic potential calculations based on the HpUreI crystal structure show negative values in periplasmic loop2 (PL2), which is part of the ph sensor, and positive values toward the center of the hexamer (Fig. 1b). The electrostatic potential is likely to pre-orient the polar urea as it enters the channel from the periplasmic side such that its NH2 moieties point towards the outside of each protomer (towards PL2) and its carbonyl oxygen points towards TMH2 near the center of hexamer. The bilayer-facing outside of the hexamer is comparatively nonpolar (Supplementary Fig. 13). Supplementary Figure 13: Electrostatic potential at the HpUreI hexamer surface computed at ph 5.3, the ph at which the crystals were grown. Side view where the surface has been colored according to the electrostatic potential computed with an adaptive PoissonBoltzmann algorithm, from -4 kt/e (red) to +4 kt/e (blue). The electrostatic potential was calculated with the program APBSmem3 at 310 K with the following dielectric constants: protein (4 ε0), membrane (2 ε0), bulk water (80 ε0). 12

13 Supplementary Table 1: Crystallographic data reductio, merging and refinement statistics Native Crystal 1 SeMet Data collection Space group P P Cell dimensions a, b, c (Å) 122.9, 122.9, , 123.3, α, β, γ ( ) 90, 90, 90 90, 90, 90 Peak Inflection Remote Wavelength (Å) Resolution (Å) R sym or R merge I / σi Completeness (%) Redundancy Refinement Resolution (Å) 3.26 No. reflections 16,314 R work / R free 23.93/29.95 No. atoms Protein 4,284 Ligand/ion 198 Water - B-factors (Å 2 ) Protein Ligand/ion Water - R.m.s deviations Bond 0.01 lengths (Å) Bond angles 1.47 ( ) *Number of xtals for each structure should be noted in footnote. *Values in parentheses are for highestresolution shell. 13

14 Supplementary Table 2: SHARP phasing statistics D min (Å) D max (Å) FOM acentric FOM centric Supplementary References 1. Thompson, J. D., Higgins, D. G., Gibson, T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673 (1994). 2. Beitz, E. TEXshade: shading and labeling of multiple sequence alignments using LATEX2 epsilon. Bioinformatics 16, 135 (2000). 3. Callenberg, K. M. et al. APBSmem: a graphical interface for electrostatic calculations at the membrane. PLoS ONE 5, e12722 (2010). 14