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• 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?

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

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

blue: monomermagenta: monomer without loop 107-120

green: 150 psmagenta: 1200 pswheat: 4500 ps

open,unliganded

• 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

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

• 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

open form (1MAW), side view open form (1MAW), top view

preTS form (1MAU), side view preTS form (1MAU), top view

• preTS => product form– KSKMS loop moves 1.3A in product crystal

structure, and 2.5A in preTS trajectories– probably separation of PPi

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)

• 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

Mg and Lys192 “share”attraction to triphosphate;holds ABD in high-energytwist

• 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

• 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)

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)