Power of Combining SAXS with Other Methods Geosynchronous Satellite - - PowerPoint PPT Presentation

Power of Combining SAXS with Other Methods

Geosynchronous Satellite Launch Vehicle ISRO

Ashish CSIR‐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?

Weaknesses Low resolution information Understanding (acceptance) is limited Sample preparation – pre‐ / post‐characterization Standards Prone to individual Strengths No need for “that” crystal or “those” NMR conditions Conditions – close to other experiments Wider range of data collection Not limited to chemical modification of the protein Reliable estimation of aggregated / non‐aggregated particle‐particle interactions globular nature or inherently disordered RG, Dmax, I0 ab initio modeling, visual insight AND THERE IS SO MUCH MORE TO EXPLORE Ways to complement/supplement

• ther biophysical data – crystallography, NMR, theoretical models ‐ templates

CD, FT‐IR, HX experiments (MS/NMR), foot printing Mutagenesis, Functional Assays, Pull‐downs A 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, Alzheimer On‐time partum – Being revised [Risk / before time partum] – 2012 JHU Gelsolin Replacement Therapy Burn & Sepsis model of mice and rat: 88% improved outcome compared to placebo ‐2008:2010:2011 Challenge Scale‐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 G3 G1 G6 G4 G5

39 133 271 137 247 419 367 511 516 618 640 731

SAXS based Insight 3 –state vs. 2‐ state?

PDB ID: 1D0N Pope & Gooch 1997 Synchrotron Foot‐printing Mark 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. Designing F‐actin Severing Competent Minimized Gelsolins

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

20 40 60 80 100 120 140 1x10

5

2x10

5

3x10

5

4x10

5

5x10

5

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

Relative Fluorescence Time (sec)

42-161 36-161 25-161 1-161 G1-G2 G1-G3 dT GSN GSN G4-G6 G2-G6 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 2000 4000 6000 8000

* **

Rate of Decrement in Fluorescence 46-60 sec 31-45 sec 0-30 sec

25‐161 28‐161

Control Placebo GSN 0.5 mg GSN 1 mg GSN 2 mg GSN 4 mg GSN 8mg

20 40 60 80 100 120 140 160

## # % of Plasma Gelsolin Levels (pGSN)

pGSN 20 40 60 80 100 120

48hours

20 40 60 80 100 120

G1-G3 GSN GSN + rGSN PBS

% of GSN levels in mice injected with rGSN (Based on Western Blot)

24 hours

Control GSN 28-161

1 2 3 4 5 6 7 25 50 75 100

Control PBS GSN G1-G3 28-161 G4-G6 G2-G6

Days Percent Survival

Current status: 8 mg dose per mice ~ 24 gm dose for 150 pound human 1 mg per mice ~ 3 gm dose!

Peddada N et al Under Review Provisional Patents Filed

G 2

• G

6 G S N d T G S N G 1

• G

3 G 1

• G

2 1

• 1

6 1 2 5

• 1

6 1 3 6

• 1

6 1 4 2

• 1

6 1

20 40 60 80 100 120

∆F [Normalized to F-actin alone]

pH 7 pH 6 pH 5 40 80 120

pH 7 pH 5

G 2

• G

6 G S N d T G S N G 1

• G

3 G 1

• G

2 1

• 1

6 1 2 5

• 1

6 1 3 6

• 1

6 1 4 2

• 1

6 1 20 40 60 80 100 120 ∆F [Normalized to F-actin alone] EGTA 1mM Ca2+ G 2

• G

6 G S N 2 5

• 1

6 1 2 8

• 1

6 1 3

• 1

6 1 3 2

• 1

6 1 3 4

• 1

6 1 2 8

• 1

5 8 2 5

• 1

5 6 40 80 120 ∆F [Normalized to F-actin alone]

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! Reabsorption Secretion Excretion Filtration is driven by hydraulic/blood pressure in the capillaries

• f the glomerulus, which in turn are formed by specialized cells

called podocytes. Podocytes have interdigitated shape known as foot processes.

Nephrin Membrane Neph1 Podocin Actin Cadherin FAT Foot process ZO-1

Interaction between Cytoplasmic domain

• f Neph1 and PDZ1

domain of ZO‐1 is somehow critical for functional shape

• f podocytes

If we can solve the structure of Neph1CD/PDZ‐1 ZO‐1, then ……..may be we can…? No sequence similarity based template 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 Concentration Unliganded proteins Molecular mass Dmax 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 1 50 15.6 ± 0.01 66 9.5 460 Sample 2 50 15.7 ± 0.01 49 7 339 Sample 3 50 15.6 ± 0.02 24 3.4 165 Sample 4 50 15.7 ± 0.07 12 1.7 82 His‐Neph1‐CD 35 Sample 1 70 21.3 ± 0.03 152 4.4 125 Sample 2 70 21.4 ± 0.05 93 2.7 77 Sample 3 70 21.4 ± 0.07 41 1.2 34 Neph1‐CD 22 Sample 1 70 18.2 ± 0.3 11 0.5 22 Sample 2 70 18.3 ± 0.6 6.5 0.3 13.6 GST‐Neph1‐ CD 53 Sample 1 110 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 reconstruction Filtering parameter and CD based fold based model

Mallik et al 2012

Complex of Neph1CD/ZO-1 PDZ1

Indirect Fourier transformation Molar ratio of His‐ZO‐1/His‐Neph1 Dmax Rg I0 Expected I0 Percentag e 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

• f

mammalian Neph proteins in Drosophila melanogaster Deciphering the molecular details

• f Neph1CD/Myo1c interaction

and determining its physiological significance The 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 Absorbance @280nm Elution Time (mL) 32 34 36 38 40

1:1 Complex Neph1-CD PDZ1 ZO-1

2 Hours 14 Hours 24 Hours 48 Hours Absorbance @280nm Elution Time (mL)

Few molecules

50 0 0 50 0 5 0 1 0 5 2 0 4 0 6 0 8 0 1 0 0

Percentage of peak area under complex

• vs. all peaks in FPLC profiles

F old d ilu tio n of X us e d (re lativ e t o m o le s o f p ro te in s ) 2 h ou rs 14 ho urs 24 ho urs 48 ho urs 60 ho urs

N O ID 50 0 0 50 0 5 0 5 2 0 4 0 6 0 8 0 1 0 0

Percentage of peak area under complex

• vs. all peaks in FPLC profiles

F o ld d ilu tio n o f X u s e d (re la tiv 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

NO BINDING OCCURS BETWEEN ∆THV Neph1-CD/ZO-1-PDZ1

Supplementing GFC data

Keeping Fingers crossed

Provisional coverage Filed

Hour-Glass Model of the Flu Infectivity

Crazy Example

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

Log10 I(Q) Log

10 Q

.0 2 .0 4 .0 6 .5 1 .0

Normalized Log I0

Q

2

Drug Site/Peptide Docking

Identification of Druggable Site: 1.Conserved in all known pathogenic strains of flu 2.In folded trimer, surface exposed 3.No propensity to undergo glycosylation 4.Involved in keeping interchain contacts Penetrating Binder Peripheral Binder

in vitro experiments

in vitro validation 1.Peptides were synthesized, purified, characterized 2.SAXS experiments were repeated 3.[Peptide]/[HA trimer] ~ 3:1

8 7 6 5 4 3 100 200 300 400 500 600

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

Maximum Linear Dimension (Å)

pH

8 7 6 5 4 3 100 200 300 400 500 600

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

Maximum Linear Dimension (Å)

pH

8 7 6 5 4 3 100 200 300 400 500 600

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

Maximum Linear Dimension (Å)

pH

8 7 6 5 4 3 100 200 300 400 500 600

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

Maximum Linear Dimension (Å)

pH

8 7 6 5 4 3 100 200 300 400 500 600

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

Maximum Linear Dimension (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…..

Weaknesses Low resolution information Understanding (acceptance) is limited Sample preparation – pre‐ / post‐characterization Standards Prone to individual Strengths No need for “that” crystal or “those” NMR conditions Conditions – close to other experiments Wider range of data collection Not limited to chemical modification of the protein Reliable estimation of aggregated / non‐aggregated particle‐particle interactions globular nature or inherently disordered RG, Dmax, I0 ab initio modeling, visual insight AND THERE IS SO MUCH MORE TO EXPLORE Ways to complement/supplement

• ther biophysical data – crystallography, NMR, theoretical models ‐ templates

CD, FT‐IR, HX experiments (MS/NMR), foot printing Mutagenesis, Functional Assays, Pull‐downs A 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 our Works