HisH - HisF Imidazole Glycerol Phosphate Synthase: regulates 5 th step histidine biosynthesis HisH...
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![Page 1: HisH - HisF Imidazole Glycerol Phosphate Synthase: regulates 5 th step histidine biosynthesis HisH class I glutamine amidotransferase HisF alpha-beta barrel.](https://reader033.fdocuments.in/reader033/viewer/2022051302/5a4d1b3a7f8b9ab05999e625/html5/thumbnails/1.jpg)
HisH - HisF• Imidazole Glycerol Phosphate Synthase:
regulates 5th step histidine biosynthesis• HisH class I glutamine amidotransferase• HisF alpha-beta barrel fold, cyclase rxn• Recently suggested hisF uses barrel as
efficient intermediate channel• Ammonia conduction, gating mechanism• Modeling activated complex requires
parameterization
P. O’Donoughue, R. Amaro, Z. Schulten, J Struct Biol, 2001.
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HisH
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HisH active site:
Catalytic triad
CYS84 – HIS178 – GLU180
HisH
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HisF
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HisF
Active site residues
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Top View of HisF
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Conserved gate residues
Form stable salt bridges
Gate diameter ~ 3 Å
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Predominantly hydrophobic
channel
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Docked Complex Crystal Structure
Douangamath et al., Structure, Feb. 2002. PDB code: 1GPW
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Glutamine binds in hisH
active site
Hypothetical Coupling Mechanism
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PRFAR binds to hisF
active site
Glutamine binds in hisH
active site
Hypothetical Coupling Mechanism
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PRFAR binds to hisF
active site
Glutamine binds in hisH
active site
Activated event
Hypothetical Coupling Mechanism
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NH3 released
in 5th reaction
Hypothetical Coupling Mechanism
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NH3 diffuses across
interface
Hypothetical Coupling Mechanism
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NH3 diffuses across
interface
Hypothetical Coupling Mechanism
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NH3 travels ~10Å to
mouth of hisF
Hypothetical Coupling Mechanism
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NH3 travels ~10Å to
mouth of hisF
NH3 passes through channel
~15Å
To participate in ImGP formation
Hypothetical Coupling Mechanism
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What is known experimentally• Crystal structures of both bacterial and
eukaryotic1 organisms (2001)
• Mutational studies involving residues of both subunits in gate and at interface2
– ARG5 and GLU46 play essential roles in rxn
• The activity of hisH is dependent on the binding of the substrate at the hisF active site1Chaudhuri et al., Structure, 2001.2Klem et al., J Bactero., 2001; Beismann-Driemeyer, J Biol Chem, 2001
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Novel function for ubiquitous fold
• Substrate channeling common in glutamine amidotransferases since coupled to second reaction requiring reactive ammonia
• Allows protected / directed travel of intermediate
• Coupling two sequential reactions increases enzymatic regulation
• First time α/β barrel proposed to be used as an intermediate channel !
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Investigating the Gate Mechanism• Gate seems closed in crystal structures• Diameter of gate 3.2Å, NH3 is ~ 2Å• Use bioinformatics to narrow the search• 2 conserved ASP’s near gate• Salt bridges could be formed between ASP98
– LYS99 and/or ASP219—ARG5• Increases diameter of gate to 6.9Å• Stable! Stay in formation for ps
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Gate at entrance of hisF barrel Crystal structures
all in closed gate conformation
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Simulated Gating Mechanisms
• Followed suggestion by Chaudhuri et al. to form a hydrogen bond between 2 strictly conserved residues at the interface: TYR138 of hisH and LYS99 of hisF’s gate
• Increased the diameter of the channel from 3.2Å to 5.8Å
• Since no experimental evidence for any gating mechanism, also simulated the closed gate
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Strictly conserved
TYR 138 of hisH
Possible gating
mechanism?
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Started with 2.4Å resolution crystal
structure
System Setup
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Started with 2.4Å resolution crystal
structure
Solvated complex with explicit waters
Minimized, equilibrated using NAMD2 and
Charmm27 force field in NPT ensemble
Theoretical Biophysics Group, K. Schulten et al.
System Setup
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Ammonia Conduction
• SMD to induce the passage of ammonia through the channel on the ns timescale
• Hamiltonian of the system becomes:( ) [ ]20 0, ( ) ( )2
kH r t r z r z vt=H + - -
• NH3 through the channel at constant v = 15Å/ns
• Analyzed the resulting trajectories, forces
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Energy Barrier of Gating Mechanism
open gate entrance ~3 kcal/mol
closed gate entrance
~25-40 kcal/mol
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Free Energy Profile in Channel
THR78 SER101 SER201
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A more realistic model• Would like to model “activated” complex• Needed to build in both substrates• hisH: covalently bound glutamine /
glutamate• hisF: substrate PRFAR• No publicly available parameters for parts
of hisH substrate (thioester) and PRFAR• Today’s lab exercise will walk you through
the parameterization of the hisH substrate
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HisH Mechanism• HisH glutamine amidotransferase• Conserved catalytic triad: C84, H178, E180
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HisH Mechanism• HisH glutamine amidotransferase• Conserved catalytic triad: C84, H178, E180
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Today’s Tutorial• VMD for introduction to the system• MOE to investigate glutamine approach to active site,
make covalent bond• MOE will make initial approximation (guesses using
pattern recognition)• Semi-empirical calculations with Spartan:
– Geometry optimization, semi-empirical– Compare results of angles, bond lengths, etc. to what MOE
guesses, experimental values– Torsional potential: dihedral drive – Minimization in NAMD2, look at results