Power of Combining SAXS with Other Methods

Geosynchronous Satellite Launch VehicleISRO

AshishCSIR‐Institute of Microbial Technology

Chandigarh INDIA

SAXS Data: Strengths, Weaknesses, Ways to Complement/Supplement

Guru Mantra??

Suspect – the problem

Prospect – weigh your chances

Approach – carve the best path

Collate – physics, chemistry, biology

Empower – self/community 

Two simple questions:

How come others get (good )results?

Can SAXS bail me out?

WeaknessesLow resolution informationUnderstanding (acceptance) is limitedSample preparation – pre‐ / post‐characterizationStandardsProne to individual

StrengthsNo need for “that” crystal or “those” NMR conditionsConditions – close to other experimentsWider range of data collectionNot limited to chemical modification of the proteinReliable estimation of   aggregated / non‐aggregated

particle‐particle interactionsglobular nature or inherently disorderedRG, Dmax, I0ab initiomodeling, visual insight

AND THERE IS SO MUCH MORE TO EXPLORE

Ways to complement/supplementother biophysical data – crystallography, NMR, theoretical models ‐ templatesCD, FT‐IR, HX experiments (MS/NMR), foot printingMutagenesis, Functional Assays, Pull‐downsA lot of reading SANS

Easy Problem: Designing Biobetters of Plasma Gelsolin

Plasma Gelsolin: Prognostic Marker of Health

Skeletal Injuries, Traumatic Brain Injury,Malaria, Arthritis, Sepsis, 2° and 3° Burn,Cystic Fibrosis, Multiple Sclerosis,Allogenic Transplantation, AlzheimerOn‐time partum – Being revised[Risk / before time partum] – 2012 JHU

Gelsolin Replacement TherapyBurn & Sepsis model of mice and rat: 88% improved outcome compared to placebo

‐2008:2010:2011

ChallengeScale‐up of Mice dose to human – 24 gm!Can we lower the dose requirement?

Gelsolin: A six domain protein which requires Ca2+ or low pH

GSN G2 G3G1 G6G4 G539 133 271137 247 419367 511 516 618 640 731

SAXS based Insight

3 –statevs.

2‐ state?

PDB ID: 1D0N

Pope & Gooch 1997

Synchrotron Foot‐printingMark Chance & group

Ashish et al 2007

Ca2+

Low pH induced shape changes in Gelsolin

Garg R et al 2011

Extension of G1 domain from other domains is essential step for F‐actin severing.

DesigningF‐actinSevering

CompetentMinimizedGelsolins

Attention please: This slide does not have SAXS data or SAXS based models

0 20 40 60 80 100 120 140

1x105

2x105

3x105

4x105

5x105

F-Actin G2-G6 GSN G1-G3 G1-G2 1-161 25-161 36-161 42-161

Rel

ativ

e Fl

uore

scen

ce

Time (sec)

42-161

36-161

25-161

1-161

G1-G2

G1-G3

dT GSN

GSN

G4-G6

G2-G6

0 1000 2000 3000 4000 5000 6000 7000

*

Rate of Decrement in Fluorescence

46-60 sec 31-45 sec 0-30 sec

25-156

25-158

34-161

32-161

30-161

28-161

25-161

GSN

G2-G6

0 2000 4000 6000 8000

*

**

Rate of Decrement in Fluorescence

46-60 sec 31-45 sec 0-30 sec25‐161

28‐161

Contro

lPla

cebo

GSN 0.5 m

gGSN 1

mg

GSN 2 m

gGSN 4

mgGSN 8

mg

0

20

40

60

80

100

120

140

160

###

% o

f Pla

sma

Gel

solin

Lev

els

(pG

SN) pGSN

0

20

40

60

80

100

120

48hours

0

20

40

60

80

100

120

G1-G3

GSN

GSN + rG

SN

PBS

% o

f GSN

leve

ls in

mic

e in

ject

ed w

ith rG

SN(B

ased

on

Wes

tern

Blo

t)

24 hours

Control

GSN28

-161 0 1 2 3 4 5 6 7

0

25

50

75

100 ControlPBSGSNG1-G328-161G4-G6G2-G6

DaysPe

rcen

t Sur

viva

l

Current status:8 mg dose per mice ~ 24 gm dose for 150 pound human1 mg per mice ~ 3 gm dose!

Peddada N et al Under ReviewProvisional Patents Filed

G2-G6

GSNdT

GSN

G1-G3

G1-G2

1-161

25-161

36-161

42-161

0

20

40

60

80

100

120

∆F

[Nor

mal

ized

to

F-ac

tin a

lon

e] pH 7 pH 6 pH 5

0

40

80

120

pH 7 pH 5

G2-G6

GSNdT G

SNG1-G

3G1-G

21-16125-1

6136

-16142-1

61

0

20

40

60

80

100

120

∆F

[Nor

mal

ized

to F

-act

in a

lone

] EGTA

1mM Ca2+

G2-G6

GSN25-1

6128

-161

30-161

32-161

34-161

28-1

5825

-156

0

40

80

120

∆F

[Nor

mal

ized

to F

-act

in a

lone

]

1mM EGTA 1mM Ca2+

∆T and G1‐G3 are Ca2+/pH independent but when we chop further….?

Is the role of SAXS over?

This work is still under progress…

Ambitious Problem: Reverting “lost” filtration ability

Filtration – 180 L per day – 7.5 L per hour!ReabsorptionSecretionExcretion

Filtration is driven by hydraulic/blood pressure in the capillariesof the glomerulus, which in turn are formed by specialized cellscalled podocytes. Podocytes have interdigitated shape known asfoot processes.

Nephrin

Membrane

Neph1

Podocin

Actin Cadherin

FAT

Foot process

ZO-1

Interaction betweenCytoplasmic domainof Neph1 and PDZ1domain of ZO‐1 issomehow criticalfor functional shapeof podocytes

If we can solve the structure of Neph1CD/PDZ‐1 ZO‐1, then ……..may be we can…?

No sequence similarity basedtemplate or biophysical characterization

X‐ray crystallography based PDBs were available (2H3M)

SWAXS data analyses from the samples of His-ZO-1-PDZ1, His-Neph1-CD, and their 1:1.2 molar mixtures.

Mallik et al 2012

Indirect Fourier transformation ConcentrationUnliganded proteins

Molecular massDmax Rg I0 mg/ml μm

Å Å kDa

Hen egg white lysozyme

44 14.2 ± 0.01 14 14.2 1a

His‐ZO‐1‐PDZ1 12.1

Sample 150 15.6 ± 0.01 66 9.5 460

Sample 250 15.7 ± 0.01 49 7 339

Sample 350 15.6 ± 0.02 24 3.4 165

Sample 450 15.7 ± 0.07 12 1.7 82

His‐Neph1‐CD 35

Sample 170 21.3 ± 0.03 152 4.4 125

Sample 270 21.4 ± 0.05 93 2.7 77

Sample 370 21.4 ± 0.07 41 1.2 34

Neph1‐CD 22

Sample 170 18.2 ± 0.3 11 0.5 22

Sample 270 18.3 ± 0.6 6.5 0.3 13.6

GST‐Neph1‐CD

53

Sample 1110 24.1 ± 0.2 15.7 0.3 5.6

I0  is  defined  as  the  intensity  of scattering  at  zero  angles,  is directly  proportional  to  the product  of  molar  concentration and  the  molecular  mass  of  the scattering  sample  and  can  be approximated by extrapolating the SAXS data to Q ∼0. 

SWAXS data based structure reconstructionFiltering parameter and CD based fold based model

Mallik et al 2012

Complex of Neph1CD/ZO-1 PDZ1

Indirect Fourier transformationMolar ratio of 

His‐ZO‐1/His‐Neph1 Dmax Rg I0Expected I0

Percentage of 1:1 binding

Å Å %

0.8 80 23.9 ± 0.05 187 205 90

1.0 80 24.0 ± 0.07 190 212 90

1.2 80 24.2 ± 0.15 197 207 95

Mallik et al 2012

Attempted Complex of Neph1CD point mutants/ZO-1 PDZ1

Mallik et al 2012

Other uses of SAXS data based filtered model of Neph1-CD

Functional  study  of  mammalian  Neph proteins in Drosophila melanogaster

Deciphering the molecular details of Neph1CD/Myo1c interaction and determining its physiological significanceThe KIN1 motif highlighted in yellow.

Helmstädter et al 2012 Arif et al  Script being composed

Now, coming back to original problem

Docking Score

Filters

32 34 36 38 40

1:1 Complex

Neph1-CD PDZ1 ZO-1

2 Hours 14 Hours 24 Hours 48 Hours

Abs

orba

nce

@28

0nm

Elution Time (mL)32 34 36 38 40

1:1 Complex

Neph1-CD PDZ1 ZO-1

2 Hours 14 Hours 24 Hours 48 Hours

Abs

orba

nce

@28

0nm

Elution Time (mL)

Few molecules

5 0 0 0 5 0 0 5 0 1 0 52 0

4 0

6 0

8 0

1 0 0

Perc

enta

ge o

f pea

k ar

ea u

nder

com

plex

vs. a

ll pea

ks in

FP

LC p

rofil

es

F o ld d i lu ti o n o f X u s e d ( re la ti v e t o m o le s o f p ro te in s )

2 h o u rs 1 4 h o u rs 2 4 h o u rs 4 8 h o u rs 6 0 h o u rs

N O I D 5 0 0 0 5 0 0 5 0 50

2 0

4 0

6 0

8 0

1 0 0

Per

cent

age

of p

eak

area

und

er c

ompl

ex

vs. a

ll pea

ks in

FPL

C p

rofil

es

F o l d d i lu ti o n o f X u s e d ( re la ti v e t o m o le s o f p ro te in s )

2 h o u r s 1 4 h o u r s 2 4 h o u r s 4 8 h o u r s 6 0 h o u r s

NO

BIN

DIN

G O

CC

UR

S BE

TWEE

N

∆TH

V N

eph1

-CD

/ZO

-1-P

DZ1

Supplementing GFC data

Keeping Fingers crossed

Provisional coverage Filed

Hour-Glass Model of the Flu Infectivity

CrazyExample

Shape of HA trimer

0.01 0.1

0.1

1

10

100 pH 8 pH 7.5 pH 6.7 pH 5.7 pH 4.7 pH 3

Log 1

0 I(Q

)

Log10 Q0.0002 0.0004 0.0006

0.5

1.0

Nor

mal

ized

Log

I 0

Q2

Drug Site/Peptide Docking

Identification of Druggable Site:1.Conserved in all known pathogenic strains of flu2.In folded trimer, surface exposed3.No propensity to undergo glycosylation4.Involved in keeping interchain contacts

Penetrating Binder Peripheral Binder

in vitro experiments

in vitro validation1.Peptides were synthesized, purified, characterized2.SAXS experiments were repeated 3.[Peptide]/[HA trimer] ~ 3:1

8 7 6 5 4 3100

200

300

400

500

600 Native HA +Peptide P1 +Peptide P2 +Peptide P3 +Peptide P4 +Peptide P5

Max

imum

Lin

ear D

imen

sion

(Å)

pH8 7 6 5 4 3

100

200

300

400

500

600 Native HA +Peptide P1 +Peptide P2 +Peptide P3 +Peptide P4 +Peptide P5

Max

imum

Lin

ear D

imen

sion

(Å)

pH8 7 6 5 4 3

100

200

300

400

500

600 Native HA +Peptide P1 +Peptide P2 +Peptide P3 +Peptide P4 +Peptide P5

Max

imum

Lin

ear D

imen

sion

(Å)

pH8 7 6 5 4 3

100

200

300

400

500

600 Native HA +Peptide P1 +Peptide P2 +Peptide P3 +Peptide P4 +Peptide P5

Max

imum

Lin

ear D

imen

sion

(Å)

pH8 7 6 5 4 3

100

200

300

400

500

600 Native HA +Peptide P1 +Peptide P2 +Peptide P3 +Peptide P4 +Peptide P5

Max

imum

Lin

ear D

imen

sion

(Å)

pH

H1N1 H5N1 H9N2 H3N2 H14N5

Patent Filed

Test Case

SAXS Data: Strengths, Weaknesses, Ways to Complement/Supplement

Guru Mantra??

Suspect – the problem

Prospect – weigh your chances

Approach – carve the best path

Collate – physics, chemistry, biology

Empower – self/community 

Two simple answers:

You will also good results….

If problem is right, and SAXS is applied correctly…..

WeaknessesLow resolution informationUnderstanding (acceptance) is limitedSample preparation – pre‐ / post‐characterizationStandardsProne to individual

StrengthsNo need for “that” crystal or “those” NMR conditionsConditions – close to other experimentsWider range of data collectionNot limited to chemical modification of the proteinReliable estimation of   aggregated / non‐aggregated

particle‐particle interactionsglobular nature or inherently disorderedRG, Dmax, I0ab initiomodeling, visual insight

AND THERE IS SO MUCH MORE TO EXPLORE

Ways to complement/supplementother biophysical data – crystallography, NMR, theoretical models ‐ templatesCD, FT‐IR, HX experiments (MS/NMR), foot printingMutagenesis, Functional Assays, Pull‐downsA lot of reading SANS

Acknowledgements

Dr. Joanna Krueger, UNC Charlotte

Dr. Deepak Nihalani, U Penn

US‐DOE beam line access

My huge & crazy team in Chandigarh‐Dr. Renu Garg – GRT translation‐Leena Mallik – Neph1‐Yogendra Rathore – CD4/gp120 , SK/HPG, t‐PA,…..‐Ashish Solanki – Neutralizing mAbs, Chip assays…‐Kalpana Pandey – Anti‐influenza‐Shikha Singh – TIR domains of TLRs‐Nagesh Peddada – Bonsai Gelsolins, PDZs, …‐Pankaj Sharma – Lysozyme amyloids, PDZs‐Amin Sagar – Actin Nucleation, CD4s, β‐lac………‐Reema – Rediscovering Calmodulin‐Maulik – Designer Antibodies‐Samir K. Nath

Project Assistants

ATSAS team –Svergun

Critics

Reviewers of Journals who declined ourWorks