J. I. Mujika

University of the Basque Country

Molecular Dynamics Simulations: an Useful Tool to Investigate the Metal Release

Mechanism from Serum Transferrin Protein

3

Theoretical Chemistry group at the University of the Basque Country

Group web page: http://www.ehu.es/chemistry/theory/

4

Outline:

Motivation of the project: 'Aluminum Age'

Mechanim of Serum Transferrin protein.

Biological role of serum transferrin (sTf) Results Conclusions

Acknowledgments

5

Part I: Aluminum Age

Aluminum in biological systems

6

Oxygen (46.6%)Silicon (27.7%)Aluminium (8.1%)Iron (5.0%)Calcium (3.6%)...

Abundance of elements in Earth's crust

% in mass

But Al(III) is not essential in the human body

7

2) Low absorption

Al(III) intake in the human body

Figure taken from Exley C. J. Inorg. Biochem. 2003, 97, 1

1) Biogeochemical control

Al(III)

Low traces of Al(III) in the human body

8

2) Aging

1) Human interventionAl(III)

Significant Al(III) concentration in the human body

water treatment pharmaceuticals drugs foodstuffs acidification of water and soil …

Al(III) intake in the human body

9

Aluminium does not present an immediate toxicity, but:

Involved in several diseases: dialysis dementia, renal

bone diseases, …

Inhibits the function of Mg or Ca metalloproteins

Related with neurodegenerative diseases, such as

Alzheimer's disease (AD) or Parkinson disease (PD)

Little information about Al(III) at molecular level

Computational chemistry can get access to this information

10

Al(III) Speciation

90% Transferrin

8% Citrate

2% Others

Percentages refer to amount of Al(III) interacting with each species

In blood

Brain extracellular fluid

Cells

Blood brain barrier

11

Part II: Serum Transferrin

Understanding the mechanism

12

● Transferrins, a family of iron-binding proteins: lactoferrin, ovotransferrin, serum transferrin.

● High sequencial similarities between them.

● Serum transferrin transports iron in blood and delivers it into cells.

● Tf is 30% saturated with iron, so binds other metals (as aluminium).

13

In serum (pH=7.4), metal load Tf is recognized by Transferrin receptor I (TFR) and introduced in cell (pH=5.5) by endocytosis.

TfMC

Tf

C

TfM

C

TFRTf

MC

TFRTf

MC

TFRTf

M

C

14

Metal binding site

15

N I

N II

ﾺ

ﾺ

X-ray structures (holo- vs apo-form)

16

Lateral view

N I

N II

Top view of N II subdomain

N II

X-ray structures (holo- vs apo-form)

Hinge bending + hinge twistingHow these global motions are modulated?

17Lys206-Lys296 interaction modulates the protein opening

”Dilysine trigger” explanation

Lys206

N I

N II

Lys296

18

Some authors(1) pointed out that dilysine trigger is insufficient to explain the domain opening

(1) H. M. Baker et al. (2007) Acta Cryst., D63, 404-414

19

(1) H. M. Baker et al. (2007) Acta Cryst., D63, 404-414

● Protonation of carbonate ion

● Protonation and posterior dissosiaction of His249

● Protonation of Tyr188 by Lys296

Some authors(1) pointed out that dilysine trigger is insufficient to explain the domain opening

20

But, this is a mere hypothesis!

● Protonation of carbonate ion

● Protonation and posterior dissosiaction of His249

● Protonation of Tyr188 by Lys296

(1) H. M. Baker et al. (2007) Acta Cryst., D63, 404-414

Some authors(1) pointed out that dilysine trigger is insufficient to explain the domain opening

Physiol. pH

Acid pH (Tyr188O-)

Systems Simulated with MM Molecular Dynamis

Mujika et al. Biochemistry (2012), 51, 7017

Acid pH (Tyr188OH(1)) Acid pH (Tyr188OH(2))

24

Technical Details

● Systems studied: Fe(III)-sTf and Al(III)-sTf

● Gromacs 4.5.3

● Initial geometry: 1A8E (iron-loaded human Tf)

● Charmm27 force field (TIP3 water molecules)

● Al(III) Lennard-Jones parameters taken from Faro et al, J. Chem. Phys. (2010), 132, 114509

● Periodic Boundary Conditions (NVT ensemble)

● Short-range electr. and VdW interactions: 14 Å Cut-off

● Long-range electrostatic calculated with PME

● Long-range dispersion correction to energy and pressure

● 1 ns equilibration + 100 ns production (time step of 2 fs)

25

MD analysis on Al(III)-sTf system

(similar conclusions with Fe(III)

Mujika et al. Biochemistry (2012), 51, 7017

26

Phys pHAcid pH (Tyr188O-)

Acid pH (Tyr188OH(1))

Acid pH (Tyr188OH(2))

27

Thr120

Arg124

Tyr95

Tyr188

Lys296

Lys206

Asp63

His249

Glu83

Tyr85

CO3

MDPhys

28

Thr120

Arg124

Tyr95

Tyr188

Lys296

Lys206

Asp63

His249

Glu83

Tyr85

CO3H

MDAcid (Tyr188O-)

Dilysine trigger does not prompt the protein opening

29

Thr120

Arg124

Tyr95

Tyr188

Lys296Lys206

Asp63

His249

Glu83

Tyr85

CO3H

(Waters not shown)

MDAcid (Tyr188OH(1))

30

Arg124

Tyr95

Tyr188

Lys296

Lys206

Asp63

His249

Glu83

CO3H

(Waters not shown)

MDAcid (Tyr188OH(2))

31Arg124 has been involved in metal release(1)

(1) T. E. Adams et al. (2003) J. Biol. Chem., 278, 6027-6033

Position of Arg124

Phys pHAcid pH (Tyr188O-)

Acid pH (Tyr188OH(1))

Acid pH (Tyr188OH(2))

32

d

N I

N II

Phys pHAcid pH (Tyr188O-)

Acid pH (Tyr188OH(1))

Acid pH (Tyr188OH(2))

33

Principle component analysis

1st Principle Component 2nd Principle Component

Technique to identify main global motions during a MD trajectory

35

Release mechanism

Lateral view Top view of N II subdomain

N II

MD structures (MDPhys

vs MDAcid (Tyr188OH(1))

)

N I

N II

36

A stepwise mechanism

1) Hinge-bending

2) Hinge-twisting

Release mechanism

38

Conclusions

MM Molecular Dynamics simulations suitable tool for exploring the sTf opening during the metal release process

The simulations show the role played by the main amino acids at different pH conditions.

Dilysine trigger insufficient to induce the conformational change

The simulations provide clear evidences for the Lys296-Tyr188 proton transfer hypothesis

The metal release may follow a two-step mechanism initialized by hinge bending

39

Acknowledgments

● Group of Theoretical Chemistry at the University of the Basque Country

● Elena Akhmatskaya and Bruno Escribano

● All of you for your attention!!