1. Computational Studies of Tryptophanyl-tRNA Synthetase: Activation of ATP by Induced-Fit Kapustina, M. and Carter, C.W. (2006) J. Mol. Biol. 362:1159-1180.

3. TrpRS - type I tRNA synthetase, 326 aa ( B. stearotherm. ) –domains: RS – Rossman fold, dinucleotide binding domain ABD – anti-codon binding domain –crystal structures open: 1MAW (ATP), 1MB2 (Trp) preTS: 1M83, 1MAU (ATP+Trp+Mg 2+ ) closed: 1I6L –conformational changes (induced fit) small-scale: KMSKS catalytic loop (107-120) large-scale: domain rotation between RS and ABD Motivating questions: stability vs. ATP affinity paradox –open-form: K d =0.4 mM ATP, 177 Å 2 exposed surface area –preTS-form: K d =~8 mM ATP, 23 Å 2 exposed surface area –why does open-form bind ATP tighter, despite the fact that preTS makes more protein-ligand interactions and is less solvent accessible? –how is induced-fit triggered? how is preTS activated?

4. Computational Studies – Molecular Dynamics Simulations advantages and disadvantages SIGMA - Jan Hermans, UNC (based on CHARMM?) –time step: 2 fs –trajectories: up to 5000 ps (5 ns) “validation”: compare mean positional RMSD to crystal B- factors –simulation under-estimates thermal motions –but there is relative correlation open, unliganded closed, liganded

5. Observations – MD simulation of open form 4 cases: unliganded, Trp, ATP, ATP+TRP all simulations were stable (no major changes) loop is flexible: accounts for 40% of RMSD –open: =107.9 –open+Trp: =87.5 –open+ATP: =80.3 opposite effects of ligand-binding on stability –Trp binds RF, reduces fluctuations in ABD –ATP binds ABD, increases fluctuations in RF

6. blue: monomer magenta: monomer without loop 107-120 green: 150 ps magenta: 1200 ps wheat: 4500 ps open, unliganded

7. preTS – unstable without (both) ligands, reverts toward open-form –unliganded and Trp-only –rearrangement over 1-2 ns ATP+Trp stabilizes preTS structure preTS+Trp+ATP – progresses toward “closed” form (like with Trp-AMP product) closed-form: stable, even without ligands

9. Characterizing domain rotations:   : hinge angle (bend), range: 10 deg  : twist (rotation), range 9 deg Table 3. Average hinge and twist rotation angles Hinge,  Twist,  OPENPreTSProductOPENPreTSProduct Initial69.9 ± 0.7062.5 ± 0.2462.3 ± 0.23−0.35 ± 0.458.9 ± 0.344.6 ± 0.12 Average over last ns No ligand70.9 ± 0.8467.2 ± 0.3663.0 ± 0.55−1.66 ± 0.952.51 ± 0.74.27 ± 0.74 +Trp71.8 ± 1.1065.80 ± 0.82ND−0.7 ± 1.362.28 ± 1.39ND +ATP69.8 ± 0.52ND 0.67 ± 1.63ND +ATP + MgND63.5 ± 0.47ND 3.80 ± 0.967ND +ATP + Trp69.6 ± 0.5262.8 ± 0.87ND1.88 ± 0.7057.05 ± 0.958ND +ATP + Trp + MgND63.0 ± 0.35ND 5.70 ± 1.110ND

11. Interactions - 4 key lysines –acceptor loop: K109, K111 –KMSKS loop: K192, K195 simulations of open form liganded with Trp+ATP show K109 in loop moves 14A to contact O in ATP triphosphate however, K111 contacts triphosphate in preTS; probably exchange coordination mutation K111A leads to rapid loss of twist in preTS – probably essential in assembly of induced fit Trp approaches ATP; may cause ordering of 107-120 loop Trp ATP Open form

12. open form (1MAW), side view open form (1MAW), top view preTS form (1MAU), side view preTS form (1MAU), top view

13. preTS => product form –KSKMS loop moves 1.3A in product crystal structure, and 2.5A in preTS trajectories –probably separation of PPi

14. Effect of Mg 2+ does not affect domain rotation of fully-liganded preTS no direct contacts with protein side-chains Mg 2+ coordinates triphosphate tail –5-coordinate sites filled, 3 by O’s, 2 by waters –unusually long bond distances: 2.54A vs. 1.85A avg preTS+ATP+Mg: –maintains  hinge, but  untwists like product state preTS+Trp+ATP+Mg: –catalytic loop pops open after 2500 ps without Mg, triphosphate uncoils, domains untwist (by 5-7 degrees)

15. unrestrained simulations: –Mg moves closer to triphosphate O’s –distrupts K111 and K192 interactions, causing loop excursions add harmonic restraints –quadratic: E =... +  i  (dist(Mg,O i )-2.54) 2 use potential of mean force (PMF) to estimate force necessary to counter tendency to move Mg –try different force constants  till achieve balance –prevent 0.7A displacement –about 5% of strength of Coulombic interaction with restraints, preTS simulations remain stable –domains do not untwist; lysines stay in contact

16. Mg and Lys192 “share” attraction to triphosphate; holds ABD in high-energy twist

17. Coupling of Mg:ATP:Lys192 interaction to domain rotations –restrain centers-of-mass with 500 kcal/mol. A 2 to prevent hinge opening and ABD untwisting –Lys192 and Lys111 stay in contact with ATP O’s

18. Model of allosteric behavior –KNF (yes) – induced fit, tertiary changes propagate –MWC (no) – symmetry effect, quartenary coupling –preTS is stable only with ligands (ATP+Mg) –increased interactions of ATP with active site compensate for strain of domain-twisting –supplies “energy” for catalysis (?) note: simulations done with monomer –negative cooperativity of dimer –also supports KNF (only one consistent with induced- fit)

20. Summary of Trp-tRNA synthetase binding and activation formation of preTS by induced-fit –ATP binds, causes domain rotation, brings K109 (RF) and K192 (ABD) together –Trp binds, causes ordering of acceptor loop –Trp brought in contact with ATP in preTS –K109 replaced by K111 –Mg binds triphosphate tail –Mg helps hold K192 and K111 in place (near triphosphate) –high Mg-O distances reflect strain in twisted state catalysis –domains untwist (partially), but do not open up (hinge angle) –PPi moves with KSMKS loop as it opens up –Mg stabilizes transition state (AMP -1 ) for transfer to Trp (acylation)