computational studies of tryptophanyl-trna synthetase: activation of atp by induced-fit
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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. TrpRS - type I tRNA synthetase, 326 aa ( B. stearotherm. ) domains: RS – Rossman fold, dinucleotide binding domain - PowerPoint PPT PresentationTRANSCRIPT
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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.
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• 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+Mg2+)• 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: Kd=0.4 mM ATP, 177 Å2 exposed surface area– preTS-form: Kd=~8 mM ATP, 23 Å2 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?
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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
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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: <B107-120>=107.9
– open+Trp: <B107-120>=87.5
– open+ATP: <B107-120>=80.3
• opposite effects of ligand-binding on stability– Trp binds RF, reduces fluctuations in ABD– ATP binds ABD, increases fluctuations in RF
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blue: monomermagenta: monomer without loop 107-120
green: 150 psmagenta: 1200 pswheat: 4500 ps
open,unliganded
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• 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
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Characterizing domain rotations: • : hinge angle (bend), range: 10 deg• : twist (rotation), range 9 deg
Table 3. Average hinge and twist rotation angles
Hinge, Twist, OPEN PreTS Product OPEN PreTS Product
Initial 69.9 ± 0.70 62.5 ± 0.24 62.3 ± 0.23 −0.35 ± 0.45
8.9 ± 0.34 4.6 ± 0.12
Average over last ns
No ligand 70.9 ± 0.84 67.2 ± 0.36 63.0 ± 0.55 −1.66 ± 0.95
2.51 ± 0.7 4.27 ± 0.74
+Trp 71.8 ± 1.10 65.80 ± 0.82
ND −0.7 ± 1.36 2.28 ± 1.39 ND
+ATP 69.8 ± 0.52 ND ND 0.67 ± 1.63 ND ND
+ATP + Mg ND 63.5 ± 0.47 ND ND 3.80 ± 0.967
ND
+ATP + Trp 69.6 ± 0.52 62.8 ± 0.87 ND 1.88 ± 0.705 7.05 ± 0.958
ND
+ATP + Trp + Mg ND 63.0 ± 0.35 ND ND 5.70 ± 1.110
ND
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• 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
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open form (1MAW), side view open form (1MAW), top view
preTS form (1MAU), side view preTS form (1MAU), top view
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• preTS => product form– KSKMS loop moves 1.3A in product crystal
structure, and 2.5A in preTS trajectories– probably separation of PPi
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Effect of Mg2+
• does not affect domain rotation of fully-liganded preTS
• no direct contacts with protein side-chains• Mg2+ 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)
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• 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,Oi)-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
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Mg and Lys192 “share”attraction to triphosphate;holds ABD in high-energytwist
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• Coupling of Mg:ATP:Lys192 interaction to domain rotations– restrain centers-of-mass with 500
kcal/mol.A2 to prevent hinge opening and ABD untwisting
– Lys192 and Lys111 stay in contact with ATP O’s
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• 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)
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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)