Cell, Volume 126

Supplemental Data

Crystal Structures of T. brucei MRP1/MRP2 Guide-RNA Binding Complex Reveal RNA Matchmaking Mechanism

Maria A. Schumacher, Elham Karamooz, Alena Zíková, Lukáš Trantírek, and Julius Lukeš

Figure S1. FP Studies of MRP1/MRP2 Interaction with Oligo II in the Presence and Absence of

5 mM Magnesium Chloride (Binding Buffer: 25 mM Tris pH 7.5, 50 mM NaCl)

These data show that magnesium is not required for high affinity binding (Kd of 6.9 ± 1.0 nM in

the presence of 5 mM Mg2+ versus 5.1 ± 0.7 nM in its absence). Without magnesium (red �);

with magnesium (blue �).

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Figure S2. FP Studies of the Salt Effect of MRP1/MRP2 Binding to the ssRNA 27-mer Adenine

Oligonucleotide

Representative normalized, binding isotherms are shown and the Kds for 50 mM NaCl (�); 100

mM NaCl (blue �); 200 mM NaCl (green �) were 32.6 ± 9.0 nM in 50 mM NaCl, 40.5 ± 6.5

nM in 100 mM NaCl and the binding was essentially eliminated at a salt concentration of 200

mM NaCl.

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Figure S3. FP Studies of Effect of RNA Conformation on MRP1/MRP2 Binding

(A) Binding isotherm of the interaction between MRP1/MRP2 and the oligo(U) tail of gRNA

after heating the RNA to 100 °C to unfold it.

(B) Binding isotherm of the interaction of MRP1/MRP2 with stem/loop I after heating the RNA

molecules to 100 °C. The binding buffer in both cases was 25 mM Tris pH 7.5, 50 mM NaCl.

Representative binding isotherms are shown with mP as the y-axis and concentration in nM of

MRP1/MRP2 “dimer” as the x axis. The Kds after unfolding for the oligo(U) tail and stem/loop I

were 26.6 ± 5.0 nM and 51.1 ± 4.1 nM, respectively.

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Figure S4. 2Fo− Fc Electron Density Map (Blue Mesh) Contoured at 1.0 σ Showing the Region

around Arg67 and Cyt19

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Figure S5. Crystal Packing of the MRP1/MRP2-gRNA Complex

Shown in green is the MRP1/MRP2-gRNA asymmetric unit (composed of one MRP1/MRP2

dimer and one gRNA molecule). Shown superimposed onto the MRP1 molecule in the

asymmetric unit is MRP2 along with its bound stem/loop II (white). Shown colored according to

atom type (yellow, blue, red and green for carbon, nitrogen, oxygen and sulfur) are the symmetry

related molecules (showing ONLY the protein) and how they pack. This figure clearly reveals

that were the stem/loop II to bind in MRP1 (white instead of green), steric clash would result

with symmetry related residue side chains Arg150, Tyr151, Pro175, and Asp188 and strand two

of stem/loop II. By contrast, binding of the single stranded, unfolded anchor sequence is readily

accommodated.