Protein Splicing: an Ancient Efficient Self-Catalytic Process€¦ · Protein Splicing In all three domains of life: archaea, bacteria and eukarya In unicellular organisms Probably

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!!