COMPUTER SIMULATION TO UNDERSTAND HOW ENZYMES WORK

Carme RoviraUniversitat de Barcelona, ICREA &

Institute of Theoretical and Computational Chemistry, Spain

What are enzymes?

Adapted from https://www.ebi.ac.uk/training/online/course/introduction-protein-classification-ebi/protein-classification

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What are enzymes?

• Most enzymes are proteins

• Enzymes serve as catalyst to accelerate biochemical reactions

• Enzymes are not changed in this process

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Saliva: amylase

Stomach:proteases

Small intestine:lipases,amylase, lactase

Picture fromwww.divavillage.com

DIGESTIVE ENZYMES

sugar units(monosaccharides)

Long carbohydrate molecule

digestion

GLYCOSIDASESCarme Rovira

Starch is our main source of energy

Starchy foodsglycosidic bonds

(≈ 1/3 of our daily food intake)

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R. Wolfenden & M. J. Snider.Acc. Chem. Res. 2001, 34, 938-945

The power of enzymes as catalysts

t1/2 = 1 billion years

t1/2 = 1 million years

t1/2 = 1 thousand years

t1/2 = 1 year

t1/2 = 1 day

t1/2 = 1 minute

age of the earth

O-glycoside hydrolysis 5-8 million years

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How enzymes work

PASS

AB

© 2010 Nature Education All rights reserved.

Without enzyme With enzyme

Ener

gy

Reaction pathway

Activationenergy

A

B

A

B

Enzymes lower the activation energyof the biochemical reaction

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How enzymes work

PASS

AB

GLYCOSIDASE

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Enzymes are very specific

Example: α-1,4-glucosidase “cuts” glycosidic bondsbetween atoms 1 and 4 of the α-glucose units of glycogen

n

1 4

...OH OH OH OH

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Lack of lactase causeslactose intolerance

GALACTOSE GLUCOSE

Enzymes and disease

Lack of α-1,4-glucosidase causes accumulation of glycogen in the liver, leading to the Pompe disease

GLYCOGEN

LACTOSE

GLUCOSE UNITS

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Knowing how enzymes work isimportant for drug design

Relenza Tamiflu

Synthesis of molecules that inhibit a disease-related enzyme

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Computer simulation to understandhow enzymes work

• Computer simulation provide molecular details of enzyme action

• Short-lived states (e.g. transition states) can be identified

• Important for inhibitor (drug) design

Reaction pathway

A

B

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Impact of High Performance Computing

• The large increase of computer power in recent years has contributed enormously to the progress of computer simulation of enzymes (computational enzymology)

• Better models and precise methods can be used

• Meaningful predictions can be made

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Clarifying mechanisms of enzymesthat process carbohydrates

Gas2 glucosidase, an antifungal target

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Simulation performed in MareNostrum (BSC)Raich et al. J. Am. Chem. Soc. 138, 3325 (2016)

Clarifying mechanisms of enzymesthat process carbohydrates Carme Rovira

(Newton’s equations)

Molecular dynamics

Initial conditions:

• Atomic positions (e.g. crystal structure): {ri}• Atomic velocities (e.g. random velocities scaled to desired T): { }iv

Motion of atoms from

ri (t)i

2i

2

rr

dEd

tddmi −=

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at t = t + ∆t

i2

i2

rr

dEd

tddmi −=

Molecular dynamics

Solve

{ } { }ii andnew vr

t = 0

{ } { }ii and:velocitiesandpositionsinitial vr

∆t ≈ 1 fs (10-15 seconds)single time step

compute forcesi

i ddEr

F −=

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Classical MD

E = Ebond + Eang + Edih + EvdW + Eelec

Energy from a force-field

{ }[ ]I(r)ρ

Rρ(r),EminE = Density Functional Theory

Molecular dynamics

Computer codes: GROMACS, NAMD, AMBER, …

Energy from quantum mechanics

Ab initio MD

Computer codes: CPMD, CP2K, QUANTUM EXPRESSO, …

Biarnés, Nieto, Planas, RoviraJ. Biol. Chem. 281, 1432–1441 (2006)

C. RoviraMethods Mol. Biol. 305, 527 (2005)

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Classical MD

E = Ebond + Eang + Edih + EvdW + Eelec

Energy from a force-field

{ }[ ]I(r)ρ

Rρ(r),EminE =

Energy from quantum mechanics

Density Functional Theory

Molecular dynamics

Ab initio MDCarme Rovira

Active center

(quantum mechanics)Rest of the system

(molecular mechanics)

Multiscale methods

“for thedevelopment of multiscale modelsfor complexchemical systems”

The quantum mechanics / molecular mechanics (QM/MM) approach

ab initio MD classical MD

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Carbohydrate degradation by glycosidases

glycosidic bondcleavage

sugar units(monosaccharides)

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Carbohydrates are very flexible

Slide from Lluís Raich(University of Barcelona)

Sugar shapes

Chair

Most abundantfor a free sugar

Envelope Boat

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Boat-likeconformation

O

Sugars change shape when they bindto enzymes

O

WHY?

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Biarnés et al. J. Biol. Chem. 2006 Biarnés et al. J. Am. Chem. Soc. 2011

Sugar distortion facilitates thebiochemical reaction

Sugars change shape when they bindto enzymes

twistedboat

O

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Biarnés et al. J. Biol. Chem. 2006 Biarnés et al. J. Am. Chem. Soc. 2011

Sugars change shape when they bindto enzymes

Sugar distortion facilitates thebiochemical reaction

Ener

gy

Reaction pathway

ActivationenergyA

B

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http://biology.tutorvista.com

Example: Golgi α-mannosidase II

Golgi complex

Cell

mannose2

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Mechanism of action

Golgi α-mannosidase II

Petersen et al. J. Am. Chem. Soc. 132, 8291 (2010)

boat

Initialcomplex

O

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Petersen et al. J. Am. Chem. Soc. 132, 8291 (2010)Williams et al. Angew. Chem. Int. Ed. 53, 1087 (2014)

Golgi α-mannosidase IIMechanism of action

anticancer inhibitors

transitionstate

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Sugar shape

• Important for inhibitor design

BUT

• The structure of the enzyme for most

glycosidase enzymes is not known

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OS2 3,OB

3S1

B1,4

1S5

B2,5

1,4B

1S3

B3,0 2SO

2,5B

5S1

4C1

OH1

OH54H5

4H32H3

2H1

Ardèvol et al. J. Am. Chem. Soc. 2010, 132, 16058

Mapping sugar shape

Metadynamics

Laio and Parrinello, PNAS 2002

mannoseEnergy map

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G. J. Davies, A. Planas, C. Rovira, Acc. Chem. Res. 45, 308 (2012).Iglesias-Fernández et al. Chem. Sci. 6, 1167 (2015).

Sugar fingerprintsEXP

glucose mannose xylose

Mapping sugar shapeCarme Rovira

Privateer software: conformational validation of carbohydrate structures Agirre, Iglesias-Fernández, Rovira, Davies, Wilson, Cowtan. Nat. Struct. Mol. Biol. 22, 833 (2015)

Structural validation

wrong conformation correct conformation

(high energy)

β α

(low energy)

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Carbohydrate synthesis

glycosidic bondformation

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Outsideof cell

Cellmembrane

Insideof cell

(cytoplasm)

virusbacteria

Sugars on the cell surface

Special Issue.March 2001: Vol. 291. no. 5512.

Carbohydratesand

Glycobiology

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Problems associated to a failure of cell-cell communication

Outsideof cell

Cellmembrane

Insideof cell

(cytoplasm)

virusbacteria

Inflammation

Autoinmune

diseases:

asthma

diabetes

allergies

multiple sclerosis

Infection

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Abnormal glycosilation: cancer

cancer cells

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• GalNAc-T2 attaches one sugar (GalNAc, a galactose derivative) to proteins

• It initiates protein glycosylation

N-acetylgalactosaminyltransferase 2 (GalNAc-T2)

GalNAc-T2

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Structure of GalNAc-T2

Catalyticdomain

Lectin domain

Linker

enzymeactive site

Collaboration with:R. Hurtado-Guerrero (U. Zaragoza)F. Corzana (Logroño)H. Clausen (Denmark)P. Bernadó (Montpellier)

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Structure of GalNAc-T2

enzymeactive site

Collaboration with:R. Hurtado-Guerrero (U. Zaragoza)F. Corzana (Logroño)H. Clausen (Denmark)P. Bernadó (Montpellier)

proteinfragment

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Metadynamics simulation

The lectin domain “catches” the peptide and brings it to the catalytic site.

Lira-Navarrete et al. Nat. Commun. 2015, 6, 6937.

Peptide binding to GalNAc-T2

Computer simulation

Atomic Force Microscopy

Lira-Navarrete et al. Nat. Commun. 2015, 6, 6937.

Computationalmicroscope

Adapted from:DOI: 10.1021/acs.jcim.5b00249(A. Magistrato)

GalNAc-T2 biochemical reaction

PEPTIDE

UDP

SUGAR

Angew. Chem. Int. Ed. 2014, 53, 8206

SUGAR TRANSFER

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hydrogen peroxide

(H2O2)

Catalase enzymes

catalase

water(H2O)

oxygen(O2)

Giorgio et al. Nature Rev. 8,722-28 (2007)

Hydrogen peroxide:• biological toxin• byproduct of metabolism

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Catalase-peroxidase (KatG)

• Antitubercular target• KatG mutations cause drug resistance to the

INH drug

Isoniazid (INH)Movie by Peter Loewen(Universty of Manitoba, Canada)

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Catalase-peroxidase (KatG)

Movie by Peter Loewen(Universty of Manitoba, Canada)

FeIV

O

Fe

M = Pt, Ru, IrYoungblood et al. J. Am. Chem. Soc. 2009, 131, 926–927D. Balcells, Adv. Org. Chem. 2016

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Summary

- Sugars have many shapes, and enzymes take advantage of this to accelerate

catalysis.

- Protein glycosylation is governed by a flexible enzyme that captures the

target protein.

- Enzymes can be redesigned to produce energy.

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Acknowledgments

Current and former group membersXevi Biarnés (IQS, Barcelona)Víctor Rojas-Cervellera (Barcelona)Albert Ardèvol (MPI, Frankfurt)Javier Iglesias-Fernández (KCL, London)Lluís Raich (UB, Barcelona)Mercedes Alfonso-Prieto (UB, Barcelona)Pietro Vidossich (UAB, Barcelona)Theoretical collaboratorsAgustí Lledós (UAB, Barcelona)Peter Reilly (Iowa State University)

Experimental collaborators:Antoni Planas (IQS, Spain)Ignacio Fita (CSIC, Spain)Peter C. Loewen (U. Manitoba, Canada)Gideon J. Davies (U. York, UK)Spencer Williams (Melbourne U, Australia)Henrik Clausen (U. Copenhaguen, Denmark)David Vocadlo (SFU, Canada)Ramón Hurtado-Guerrero (BIFI, Spain)