peterjb@bii.a-star.edu.sg Peter J. Bond (BII)

Computational modelling of host–pathogen interactions:

from atoms to systems

```

``` ```

Immune Recognition of Bacterial LPS

2

à active as dimer

• Remarkablyflexiblecavity,assessedusingtrj_cavity…Paramoetal,JCTC,2014.• “Clamshellmotions”enableadaptationofcavityvolumetodifferentligands.• Sizeofhydrophobiccomponentoflipidscorrelates(>98%)withMD-2cavityvolume.• Paramoetal.JBiolChem(2013)288:36215-

MD-2 Co-Receptor “Gauges” LPS Structure

Agonist Antagonist

Dynamicchangesinvolumedetected by trj_cavity –JustAccepted,JCTC

3

MD-2 β-Cup is a Dynamic “Clamshell”

4

F126A mutant – LPS chemical shifts perturbed J Biol Chem. (2012) 287:16346-.

•  Paramo et al. J Biol Chem (2013) 288:36215- •  Ortiz-Suarez & Bond, Structure (2016) 24:200- •  Paramo et al. Scientific Reports (2015) 5:17997 •  Berglund et al. Prog. Biophys. Mol. Biol. (2015) 119:72-

LPSbacterium

accessoryproteins

CD14TLR4

MD-2

signal

cytoplasm

TLR4: Part of a “Funneled” System?

5

à Can we use multiscale modelling to assess the “thermodynamic funnel”

hypothesis

Coarse-Grained Model for LPS & Receptors

•  Iterative parameterization based on all-atom simulations. •  Membrane lipids (angles/dihedrals) & proteins (ENMs). 6

Careful Validation of CG Lipid Model… distance

7

•  Electron density profile + cation “cross-links”. •  AC 0.255 ± 0.004 nm2 (X-ray diffraction = 0.26 nm2.) •  Dl ~4 x 10-9 (2-3 x 10-9 cm2s-1 from fluorescent labelling.) •  Free-energies of LPS affinity from atomistic simulations

(250-300 kJ mol-1) - Scientific Reports (2015) 5:17997

•  human TLR4 + MD-2 (based on X-ray structure)

•  + modelled

transmembrane helix (guided by CD experiments)

•  + POPC membrane

TM TLR4/MD-2 – Coarse-Grained Model

8

TM CD14 – Coarse-Grained Model

•  human CD14 (based on X-ray structure)

•  + GPI anchor

•  + LPS (based on atomistic assembly simulations)

•  + POPC membrane

9

CD14�LPS

ΔΔG +150 kJ/mol

LPS

10

MD2�LPS

ΔΔG -50 kJ/mol

TLR4− MD2�LPS

ΔΔG -250 kJ/mol

TLR4-MD-2 / CD14 Assembly & Lipid Exchange

•  Multiple replicas map interaction interface & dynamics. •  Assembled “productive” states seed lipid transfer sims.

*

Ryu et al. 2017, Immunity 46, 1–13.

11

LPS Multiscale Modelling: Now & Next

•  Anti-bacterial/anti-LPS/anti-CD14: thrombin fragments in wound healing. •  Schmidtchen (LKCMed, Lund Uni), Huber, et al. 2017, PNAS.

•  TLR TM domains (& peptidomimetics). Hubert Yin (U. Colorado at Boulder), Kargas, Marzinek, Holdbrook et al, 2017, BBA Biomembranes.

•  LPS/TLR4 relay – from atoms to systems via multiscale simulation, integrative modelling, and experimental calibration.(Huber et al. 2017, under review).

12

peterjb@bii.a-star.edu.sg Peter J. Bond (BII)

Computational modelling of host–pathogen interactions:

from atoms to systems

```

``` ```

Dengue Danger: Local & Worldwide

•  ~400m cases per year •  Mosquito borne (A. aegypti)

•  Different serotypes…. •  Vaccines <100% effective

14

pH induced transitions

Antibody binding

Virus maturation

All-atom MD Coarse grain MD

Ultrafast X-Ray NMR Fluorescence Spectroscopy TEM, CryoEM HXMS, SAX, WAXS

Multiscale Dynamics of Dengue (DENV)

•  “An integrated computational and experimental platform to study multi-scale dynamics”.

•  SGD$19M MoE Tier 3 project involving ~15 PI’s.

•  NUS, NTU, Duke-NUS, & BII. •  Integration of experimental

data: -  CryoEM -  H/D exchange -  SAXS -  NMR -  Fluorescence

•  Multiscale computational platform:

-  (BII, A*STAR).

15

Dengue (Flavivirus) Architecture

I. Viral envelope membrane structure / dynamics

II. Nucleocapsid architecture / interactions

16

E(TM)

DIII DI DIII

I. Modelling Viral Envelope Structure & Dynamics

DENV-2, cryoEM, PDB: 3J27

PC:PE:PS, 6:3:1 ratio (lipidomics)

III

I II

FP

Marzinek et al. (2016) Structure, 24:1410- 17

Coarse grained model based - Martini. ~4:1 mapping; particles mimic polarity / charge / H-bonding.

MultiscaleModellingoftheDengueEnvelope

CG elastic network model for envelope protein tuned to atomistic MD simulations.

Huber et al. (2016). Prog. Biophys. Mol. Biol. 18

t = 0 ns:correlation

= 0.37

PC:PE:PS (6:3:1) lipid vesicle membrane

90 E dimers + 90 M dimers

~1millioncoarse-grainedparticles(hundredsofnsrequiredforequilibration).

ComparingTheory&Experiment

Correlation between aligned electron-density grid maps, calculated as mean cosine similarity.

19

1 µs vs cryo-EM map

RefinementofEnvelope:BasisforCurvature

0%

100%

0 µs E:1 µs M:1 µs

PS

PE

PC

PC:PE:PS (6:3:1) enrichment TM & curvature require anionic lipid

Marzinek et al. Pushing the Envelope: Dengue Viral Membrane Coaxed into Shape by Molecular Simulations. (2016) Structure, 24:1410-1420.

20

“smooth”vs“bumpy”virion

IntegrativeModellingofVirion“Breathing”

FRET-envelopeexpansion.(LimXXetal.NatCommun.20178:14339.) 21

FibriansahGetal.JVirol(2013)87:7585-

•  13.7ÅcryoEM:noTM/stem/lipid.•  MD-flexiblefitting(MDFF)–external

potentialfromcryo-EMdensitymapdefinedongridofallRparticlecoords.

•  Mass-weighted(ωj)&scaled(wj–e.g.excludeTM)forceproportionaltogradientofdensitymapapplied:

Etotal=EMD+EMAP+ESS UMAP (R) = ω jVMAP (rj )j∑ Fj

MAP = −∇UMAP (R) = −wj

∂VMAP (rj )∂rj

•  Emergenceofprotein/lipidpores–ionbinding?

VirionDynamics:From“Breathing”toFusion…

•  Lateendosomalmembranemodel-PC,PE,sterols,phosphatidyl-inositol3-phosphate(PI3P)&bis(monoacylglycero)phosphate(BMP).

•  Nosignificantfusionforexpandedvirion-particleis“robust”.•  NeedformajorconformationalchangesinducedbypHdrop…

DengueVirusLifeCycle|HHMI'sBioInteractive,2010

Integrative Model of a “Spiky” Virion

iterativetestingofvesiclemodels

fittingtocryoEM(26Å)

Stem

TM

M-protein C >1.5

>0.5 <0.5

Deuterium differences

stemflexibility(HDX-MS)

viralmembrane

ectodomain(X-ray) stem+TM(cryoEM)

23

FITC-labelled peptide diffusion

Rhodamine-labelled lipid diffusion

2017. Marzinek et al. Submitted 2016 Marzinek et al, Sci Rep. 5, 19160

FP – lipid bilayer

ΔG

Membrane “Attack” by Virion Spikes

24

FP – lipid bilayer

�G

≈3�G

≈9�G

ΔG

Membrane “Attack” by Virion Spikes

25

cooperative FPs

Spiky Virus Sculpts Host Membranes

Guietal.J.Virology.90:6948-

26

q  General approach to enveloped viruses à dynamics & fusion. q  Multiscale integrative modelling of nucleocapsid. q  Future: therapeutics, antibodies…

MultiscaleFlavivirusDynamics:Now&Next

Capsid inh ib i t ion. . . Cf . Faust ino et a l . Understanding Dengue Virus Capsid Protein Disordered N-Terminus and pep14-23-Based Inhibition.‘15 ACS Chemical Biology 10:517-526.

Screening for cryptic pockets, antivirals (Soni et al., submitted, 2017).

Structural basis for antibody-induced dengue maturation / infectivity. (Wirawan et al. Submitted, 2017).

27

SGD$19M,MoETier3grant.-  Singapore-wideproject

involving15PI’s,hostedbyNUS(PaulMatsudaira).

-  ChandraVerma(BII)-  SheemeiLok(Duke-NUS)-  ThorstenWohland(NUS)-  GaneshAnand(NUS)-  GerhardGruber(NTU)

Acknowledgements

Nucleocapsidstructure:-  RolandHuber(A*STARYIG),

YueWan,AdeleneSim.-  IvoMartins,AnnaMartins

(EMBO)

Jim Warwicker Bob Ford Martin Ulmschneider Olivera Francetic Tom Piggot Syma Khalid “LPS Network” Artur Schmidtchen & Jitka Petrlova Graeme Lancaster Clare Bryant Sebastian Hiller Hubert Yin LKCMedicine & Lund BakerIDI Melbourne Uni. Cambridge Uni. Basel / EMBL Uni. Colorado Computing BII ACRC NSCC

peterjb@bii.a-star.edu.sg

Jan Marzinek Roland Huber Daniel Holdbrook Aishwary Shivgan Stefan Ivanov

Ana Martins Priscilla Boon Abhishek Soni Sonal Pai Melanie Koh