A conserved P-loop anchor limits the structural dynamics that mediate. nucleotide dissociation in EF-Tu.

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1 Supplemental Material for A conserved P-loop anchor limits the structural dynamics that mediate nucleotide dissociation in EF-Tu. Evan Mercier 1,2, Dylan Girodat 1, and Hans-Joachim Wieden 1 * 1 Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, AB T1K 3M4, Canada 2 Current address: Department of Physical Biochemistry, Max-Planck Institute for Biophysical Chemistry, Göttingen, Germany

2 FIGURE LEGENDS Figure S1. Eyring plots of mant-gtp dissociation from EF-Tu variants. Mant-GTP dissociation rate constants (k off ) were determined at different temperatures. Data points represent rates obtained from fitting individual traces in replicate experiments; wild type EF-Tu ( ), H22G ( ), M112G ( ), M112A ( ), M112L ( ). Figure S2. The P-loop forms a helical turn in simulation of EF-Tu apo M112A. P-loop backbone conformations are shown for wild type (A&F), H22G (B&G), M112G (C&H), M112A (D&I), and M112L (E&J) after 10ns of simulation of EF-Tu GTP (A-E) or EF-Tu apo (F-J). Figure S3. Interactions between the two N-terminal P-loop amino acids and helix C are conserved in P-loop NTPases. Multiple sequence alignments of the G-proteins EF-Tu, EF-G, FtsY, Ffh, Ras, Rac1, and Ran as well as the ATPases adenylate kinase and MutS were performed in ClustalW 1,2. The P-loop (top) and helix C (bottom) alignments are shown for each protein with amino acids coloured according to conservation (black: 100%, grey: >80%, light grey: >60%). Interactions between amino acids of the P-loop and helix C, shown as dashed lines, were identified in crystal structures of each G-protein bound to a non-hydrolyzable GTP analogue. The PDBIDs for the structures investigated were as follows: EF-Tu:1EFT, EF-G: 2BV3, FtsY: 2Q9B, Ffh: 2CO4, Adenylate Kinase: 1AKE, MutS: 1E3M, Ras: 3L8Z, Rac1: 1MH1, Ran: 1IBR. Interacting amino acids were defined by close approach (<3.5Å; <3.8Å for FtsY and MutS) of heavy atoms. Figure S4. Root mean-squared deviation (RMSD) of backbone atoms in EF-Tu during molecular dynamics simulations. (a) Comparison of variability of EF-Tu simulations with GTP Mg 2+ bound in the nucleotide-binding pocket. Based on this, simulations (10ns) were performed with (b) and without (c) GTP Mg + bound in the nucleotide-binding pocket of EF- Tu WT and respective variants. Each simulation was carried out with a 0.5 fs time step at 300K in an NPT ensemble; conformations were sampled every 0.5ps for RMSD analysis. The traces shown are wild type (blue), H22G (black), M112L (red), M112G (green), and M112A (purple) EF-Tu Figure S5. Relative total energies of EF-Tu during 10 ns molecular dynamics simulations. Comparison of EF-Tu WT and variants (a) free and (b) with GTP Mg 2+ bound in the nucleotidebinding pocket.

3 FIGURES Figure S1. Figure S2.

4 Figure S3.

5 Figure S4.

6 Figure S5. a b

7 Table S1. Distances between N-H bonds of the P-loop backbone and oxygen atoms of GTP phosphates based on different MD simulation segments. Distance EF-Tu wt GTP EF-Tu wt GTP EF-Tu wt GTP EF-Tu EF-Tu Measured 6-10ns 6-40ns 10-40ns H22G GTP H22G GTP 6-20ns N-H O γ2 (2.3 ± 0.3) Å (2.3 ± 0.3) Å (2.3 ± 0.3) Å (1.96 ± 0.21) Å (1.97 ± 0.20) Å N-H O β1 (3.1 ± 0.3) Å (3.0 ± 0.3) Å (3.0 ± 0.3) Å (1.97 ± 0.17) Å (1.96 ± 0.17) Å N-H O β1 (2.19 ± 0.19) Å (2.18 ± 0.19) Å (2.18 ± 0.19) Å (1.97 ± 0.19) Å (2.04 ± 0.23) Å N-H O α3 (2.63 ± 0.23) Å (2.61 ± 0.23) Å (2.61 ± 0.23) Å (2.55 ± 0.25) Å (2.48 ± 0.25) Å N-H O β1 (1.97 ± 0.14) Å (1.98 ± 0.14) Å (1.98 ± 0.14) Å (2.01 ± 0.15) Å (2.03 ± 0.15) Å N-H Thr25 O β2 (1.95 ± 0.12) Å (1.95 ± 0.12) Å (1.95 ± 0.12) Å (1.98 ± 0.12) Å (1.98 ± 0.13) Å Table S2. Probability of P-loop amino acids undergoing backbone conformational changes during steered MD. Probabilities (in percent) were computed based on the fractions of SMD simulations in which the respective amino acid occupied multiple backbone conformations. Amino Acid wt H22G M112G M112A M112L Gly His Val Thr

8 Table S3. Summary of hot P-loop amino acids in MD simulations of all EF-Tu variants. Amino acids listed for SMD were hot in 20% or more of SMD simulations. wild type H22G M112G M112A M112L K D /nm a 70 ± ± ± ± ± 100 k 20 C / s -1 b ± ± ± ± ± TΔS 20 C b /kjmol ± 1-29 ± 2-45 ± 5-44 ± 3-40 ± 1 EF-Tu GTP hot amino acids Gly18 His19 Gly18 His19 SMD hot amino acids EF-Tu Apo hot amino acids Gly22 Gly22 a b computed from kinetic analysis of the EF-Tu GTP complex computed for EF-Tu GTP dissociation References 1 Thompson, J. D., Higgins, D. G. & Gibson, T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionspecific gap penalties and weight matrix choice. Nucleic Acids Res. 22, (1994). 2 Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, (2007).