Protein Splicing: an Ancient Efficient Self-Catalytic Process
J. I. Mujika
University of the Basque Country and Donostia International Physcis Center
DNA
RNA
Protein
Intein
Intein
C-extein
C-extein
N-extein
N-extein
Transcription
Translation
Protein Splicing
(inactive)
(active)
Protein Splicing
● In all three domains of life: archaea, bacteria and eukarya
● In unicellular organisms
● Probably an ancient evolutionary origin
● Catalyzed by amino acids located in the intein (and the first one of C-extein).
● Not coenzyme or external source of energy required.
Intein
● More than 200 inteins are known ( size of 134-650 amino acids)● Four type of inteins:
a) Maxi-inteins:
Splice-domain Endonuclease domain
a) Mini-inteins:
Splice-domain
c) Ala-inteins: Ala1 instead of Cys1 or Ser1
d) Trans-splicing:
C-inteinN-intein+Protein A Protein B
Intein classification
- Protein purifications- Protein ligation- Protein labeling- ...
SplicingHost protein
SplicingForeign protein
Applications in protein engineering strategies:
a) Protein transferability
trans-SplicingAssociation
b) Timing control
InteinN-extein C-extein
Inteins show relatively low sequential similarity
CysSer(Ala)
CysSerThrAsn
B F G
Residues located in blocks B, F and G catalyze N- and
C-terminal junction cleavage
Several conserved amino acids● Directly involved in the mechanism● Catalytic role at a certain step
Mechanism: independent but coordinated 4 steps
Step 1: N-S/O acyl shift
Precursor
Linear ester
(N cleavage)
Mechanism: independent but coordinated 4 steps
Step 2:transesterificationLinear ester
Branched interm.
Mechanism: independent but coordinated 4 steps
+Step 3: C cleavage
Branched interm.
Mechanism: independent but coordinated 4 steps
Step 4: S/O-N acyl shift
+
Our research focuses on:
Step 1: N-S/O acyl shift
Precursor
Linear ester
(N cleavage)
+Step 3: C cleavage
Branched interm.
Kinetic data
N-terminal cleavage 25.6 kcal/mol
C-terminal cleavage 24.9 kcal/mol
Mills et al. J. Biol. Chem. (2005), 280, 2714
N-terminal cleavage 1 10-4 s-1
C-terminal cleavage 2.8 10-4 s-1
Applying Eyring equation:
Methodology
1. Molecular Dynamics Simulations:
- Equilibrate the system- Investigate histidines' protonation state
● Gromacs 4.5.3● Charmm27 force field● Two AAA segments added as C-extein and
N-exteins● Periodic Boundary Conditions● NVT ensemble● Equilibration: 2ns● Production of 30 ns
Initial structure: recA mini-intein from Mycobacterium tuberculosis (2IN0 pdb code)
Methodology
2. Potential Energy Surface (PES) characterization:
- QM/MM scheme- Locate stationary points along a reaction pathway- Explore alternative reaction coordinates- Methodology testing- High dependence on the initial conformation
● Charmm program● QM/MM scheme● QM part: SCCDFTB● MM part: Charmm27● Link atoms at the QM boundaries
Methodology
3. Potential of Mean Force (PMF):
- QM/MM scheme- Free energy surface characterization- Time consuming calculations- A better sampling- Reaction coordinates based on PES
● Charmm● SCCDFTB/Charmm27● Link atoms at the QM boundaries● Each window: 10 ps equilibration + 30 ps production● Free energy profile computed with WHAM
Step1: N-S/O acyl shift
Step 1: N-S/O acyl shift
Precursor
Linear ester
(N cleavage)
InteinN-extein C-extein
CysSer(Ala)
CysSerThrAsn
B F G
TXXH D
Du et al. JACS (2011), 133, 10275
2IN0 X-ray crystal structure
5.7
5.0
Asp422
His73
Cys1
pKa=6.1
pKa=7.3
Mechanism 1: activation of Cys1 side chain by His73Mechanism 2: activation of Cys1 side chain by Asp422
Path 1a Path 1b
Step 1
Step 2
QM(SCCDFTB)
MM(CHARMM27)
Product
Activated react.
Reactant
● Stepwise mechanism● 1st step: Cys1 side chain activation● 2nd step: protonation of N by His75
r.c.1: Cys1 activation by Asp422r.c.2: N protonation by His73
PES characterization to determine suitable reaction coordinates
r.c.1: Cys1 activation by Asp422r.c.2: N protonation by His73
● Stationary points at similar positions● Lower relative energy values,
specially for the second step
Comparison with higher level of theory
● QM contribution recalculated: single-point calculations at B3LYP/6-31+G(d)/CHARMM27
● Gaussian03
React
TS1
TS2
Act. react
Prod.
SCCDFTB//CHARMM27
B3LYP/6-31+G(d)//CHARMM27
PMF for Path 1a: Cys1 side chain activation by His73
Reactant (0.0)
Activated react.(7.6)
TS1(22.5)
Final snapshot
PMF for Path 1b: Cys1 side chain activation by Asp422
Reactant (0.0)
Activated react.(2.6)
TS1(8.2)
Final snapshot
PMF for Second step
Product(21.7)
TS2(35.9)
Activated react.(2.6)
Final snapshot
Step3: C-terminal cleavage
InteinN-extein C-extein
CysSer(Ala)
CysSerThrAsn
B F G
H
+Step 3: C cleavage
Branched interm.
H
But, which is the protonation state of the two histidines?
pKa=6.3
pKa=8.9
Du et al. JACS (2009), 131, 11581
Nine molecular dynamics simulations considering the three protonation states for His429 and His439
+
● In eight out of nine of the MD simulations conformation b)● Conformation a) only in one MD● Conformation a) more suitable for the reaction
Reaction mechanism for C-extein/intein cleavage
● The protonation of peptide bond N by His439 does not lead to the final product● Therefore, three steps:
1 Activation of Asn440 side chain2 Attack of Asn440 side chain at the peptide bond C3 Protonation of peptide bond N
21
3
QM(SCCDFTB)
MM(CHARMM27)
Reactant (0.0)
Activated react.(13.8)
TS1(16.8)
Final snapshot
PMF for Step1: Asn440 side chain activation by His429
Final snapshot
Interm. (15.9)Activated react.
(13.8)
TS2(19.8)
● Not possible to characterize the free energy profile for the protonation of N by His439.
● We hypothesize that another residue act as an acid. The protonated Asp422?
Last step: peptide bond cleavage
PMF for Step2: attack of Asn440 side chain at peptide bond C
N-cleavage C-cleavage
Nucleophile Cys1 Asn440
Base residue Asp422 His429
Acid residue His73 Asp422??
Main role of His Protonate N Stabilize oxyanion
His429
His439
Asn440
His73
Asp422
Cys1
Step1: N-terminal cleavage Step3: C-terminal cleavage
Conclusions
● Similar acid-base mechanism for the N- and C-terminal cleavage.● N-terminal cleavage: Asp422 seems a better base group than His73.● Asp422 stabilizes the negative charge formed at peptide bond O.● C-terminal cleavage: His429 activates the Asn440 side chain.● Different roles for the two histidines:
● His73 protonates the peptide bond N atom.● His339 stabilizes the oxyanion's negative charge.
● The SCCDFTB/CHARMM27 scheme provides reliable structures, although may overestimate the energies..
Mujika et al. J. Phys. Chem. B (2009), 113, 5607Mujika et al. Org. Biomol. Chem. (2012), 10, 1207
Acknowledgments
All of you for your attention!!