Assigning Protonation States to Residues in a Protein

Overview

Teaching: 30 min
Exercises: 10 min

Questions

• Why titratable aminoacid residues can have different protonation states?

• How to determine protonation state of a residue in a protein?

• What are the weaknesses of fixed protonation state simulations?

Objectives

• Understand why it is necessary to assign the correct protonation state

• Learn how to determine protonation state of a protein

• Learn how to assign protonation state to a residue

It is important to consider amino acid protonation states

• The protonation pattern of proteins is crucial for their catalytic function and structural stability.
• Numerous MD simulation studies have demonstrated the importance of protein protonation states [1, 2, 3, 4, 5].

How to Determine Protonation States of Residues in a Protein?

For predicting the pKa values of protein residues, several web servers and standalone programs are available.

• H++. Continuum electrostatics model
• PROPKA3.0. Empirical pKa prediction.
• PlayMolecule-ProteinPrepare. Based on PROPKA3.
• PDB2PQR. Solves Poisson-Boltzmann equation.
• MCCE. Takes into account conformational flexibility. More accurate and more challenging to use.
• PKAD. A database of experimentally determined pKa values.

There is no perfect pKa prediction method. Deviations from experimental values can sometimes be significant. The best practice is to compare the results obtained from different techniques and, if possible, to use experimentally measured values.

Calculating pKa’s

1. Calculate pKa’s of residues in the PDB entry 1RGG using H++ server.
2. What protonation states of Asp79 and His53 are appropriate for simulation at pH 6?
3. Repeat calculations using PDB2PQR server and compare the results.
4. Compare calculated pKa’s with the experimental. How accurate are the predicted pKa values?

What protonation states are appropriate for simulating Asp79 and His53 at pH 6?

Solution

If pKa > pH the probability that the residue is protonated is > 50%, and we use the protonated form.
If pKa < pH the probability that the residue is protonated is < 50% and we use the deprotonated form.

ASP79 has pKa 7.2 (experimental 7.37), it is protonated at pH 6 and we rename it to ASH
HIS53 has pKa 8.3 (experimental 8.27), it is also protonated at pH 6 and we rename it to HIP

How to select protonation state of a residue?

Assigning protonation states in structure files

• To change the form of an amino acid, change its name in the structure file

LYS (+1) - LYN  (0)  
ASP (-1) - ASH  (0)  
GLU (-1) - GLH  (0)  
HIS  (0) - HIE  (0)  
HIS  (0) - HID  (0)  
HIS  (0) - HIP (+1)    

Selecting protonation states with check_structure.

Let’s change ASP20 and ASP26 in the file 1ert.pdb to the neutral form ASH.

cd ~/workshop_amber/example_02
check_structure -i 1ert.pdb -o 1ert_protonated.pdb \
command_list --list \
"add_hydrogen --add_mode list --list A:asp20ash,A:asp26ash; \
water --remove yes"

You can verify that residues are changed by grepping ASH.

Selecting protonation states with VMD.

Change ASP20 and ASP26 in the file 1ert.pdb to the neutral form ASH and remove water.

Solution

ml StdEnv/2023 vmd
vmd

mol new 1ert.pdb
set s [atomselect top "resid 20 26"]
$s set resname ASH
set s [atomselect top "protein"]
$s writepdb 1ert_protonated.pdb
quit

Assigning protonation states with the GROMACS pdb2gmx module

• By default, pdb2gmx will select charged forms of LYS, ASP, or GLU
• For HIS, it will try to place the proton optimally

Downsides:

• Interactive
• Residue names are changed only in the topology

Limitations of Fixed Protonation State Simulations

• Difficult to understand proton-coupled conformational dynamics.
• Consider using constant pH simulations if proton-coupled dynamics are essential to your research.

Combining all structure preparation steps in one check_structure script

cd ~/workshop_amber/example_03
check_structure -i 1rgg.pdb -o 1RGG_chain_A_prot.pdb \
    command_list --list "\
        chains --select A;\
        add_hydrogen --list A:his53hip,A:asp79ash --add_mode list;\
        altloc --select A5:B,A54:B,A6:A,A13:A,A42:A,A85:A,A91:A;\
        getss --mark all;\
        ligands --remove all;\
        water --remove yes"

You can also save commands in a file and pass it as an argument to “command_list –list”:

check_structure -i 1rgg.pdb -o 1RGG_chain_A_prot.pdb command_list --list prep_1rgg.chk

References

1. Constant-pH Molecular Dynamics Simulations for Large Biomolecular Systems

2. GPU-Accelerated Implementation of Continuous Constant pH Molecular Dynamics in Amber: pKa Predictions with Single-pH Simulations

Combining all structure preparation steps in one VMD script

Combine all previous steps together and create VMD script to prepare MD simulation system for the hydrolaze PDB structure 1RGG. The script should perform the following steps:

1. Select molecule A
2. Remove non-protein molecules
3. Select location ‘B’ for residues 5, 54 and location ‘A’ for all other residues with alternative locations
4. Protonate Asp79 and His53
5. Rename CYS 7 and 96 into CYX (cross-linked cystein)
6. Save the resulting structure as 1RGG_chain_A_prot.pdb

Solution

cd ~/workshop_amber/example_03

Save the following commands in a file, e.g. prep_1RGG.vmd

# Load 1rgg.pdb into a new (top) molecule
mol pdbload 1rgg
# Select and save all chain A protein atoms
set s [atomselect top "protein and chain A"]
$s writepdb 1RGG_chain_A.pdb
# Delete the top molecule
mol delete top
# Load chain A into a new molecule 
# Loading only one chain will simplify selections commands 
mol new 1RGG_chain_A.pdb
# Protonate ASP79
set s [atomselect top "resid 79"]
$s set resname ASH
# Protonate HIS53
set s [atomselect top "resid 53"]
$s set resname HIP
# Rename cross-linked cysteins
set s [atomselect top "resid 7 96"]
$s set resname CYX
# Select the base and the alternate locations
set s [atomselect top "(altloc '') or (altloc A and resid 6 13 42 85 91) or (altloc B and resid 5 54)"]
# Save the selection
$s writepdb 1RGG_chain_A_prot.pdb
quit

Execute the script

vmd -e prep_1RGG.vmd

Key Points

• Assigning correct protonation states of aminoacids in proteins is crucial for realistic MD simulations

• Conformational changes in proteins may be accompanied by changes in protonation pattern.