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. 3621159-1180.

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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 (ATPTrpMg2)
• 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 Kd0.4 mM ATP, 177 Å2 exposed surface
  area
• preTS-form Kd8 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
open, unliganded

• 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

closed, liganded
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Observations MD simulation of open form

• 4 cases unliganded, Trp, ATP, ATPTRP
• all simulations were stable (no major changes)
• loop is flexible accounts for 40 of RMSD
• open ltB107-120gt107.9
• openTrp ltB107-120gt87.5
• openATP ltB107-120gt80.3
• 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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open, unliganded
green 150 ps magenta 1200 ps wheat 4500 ps
blue monomer magenta monomer without loop
107-120
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• preTS unstable without (both) ligands, reverts
  toward open-form
• unliganded and Trp-only
• rearrangement over 1-2 ns
• ATPTrp stabilizes preTS structure
• preTSTrpATP progresses toward closed form
  (like with Trp-AMP product)
• closed-form stable, even without ligands

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Characterizing domain rotations a, g

• a hinge angle (bend), range 10 deg
• g twist (rotation), range 9 deg

Table 3. Average hinge and twist rotation angles
Hinge, a Hinge, a Hinge, a Twist, b Twist, b Twist, b
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 TrpATP
  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
Open form
ATP
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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 gt 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 Os, 2 by waters
• unusually long bond distances 2.54A vs. 1.85A
  avg
• preTSATPMg
• maintains a hinge, but g untwists like product
  state
• preTSTrpATPMg
• 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 Os
• distrupts K111 and K192 interactions, causing
  loop excursions
• add harmonic restraints
• quadratic E ... wSi (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 w 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-energy twist
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• Coupling of MgATPLys192 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 Os

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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 (ATPMg)
• 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)