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 Weaknesses Two simple questions: Low resolution information Understanding (acceptance) is limited How come others get (good )results? Sample preparation – pre ‐ / post ‐ characterization Standards Prone to individual Can SAXS bail me out? Strengths No need for “that” crystal or “those” NMR conditions Guru Mantra?? Conditions – close to other experiments Wider range of data collection Suspect – the problem Not limited to chemical modification of the protein Reliable estimation of aggregated / non ‐ aggregated Prospect – weigh your chances particle ‐ particle interactions globular nature or inherently disordered Approach – carve the best path RG, Dmax, I0 ab initio modeling, visual insight AND THERE IS SO MUCH MORE TO EXPLORE Collate – physics, chemistry, biology Ways to complement/supplement Empower – self/community other 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 Ca 2+ or low pH 137 39 133 247 271 367 419 511 516 618 640 731 G1 G2 G3 G4 G5 G6 GSN SAXS based Insight 3 –state vs. 2 ‐ state? Synchrotron Foot ‐ printing Mark Chance & group PDB ID: 1D0N Pope & Gooch 1997 Ashish et al 2007
Ca 2+
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 5 G2-G6 46-60 sec 5x10 F-Actin 46-60 sec Relative Fluorescence G2-G6 25 ‐ 161 31-45 sec G2-G6 31-45 sec GSN G4-G6 0-30 sec GSN 0-30 sec G1-G3 GSN 5 4x10 25-161 * G1-G2 dT GSN 1-161 28-161 ** 25-161 G1-G3 5 3x10 36-161 30-161 G1-G2 42-161 32-161 1-161 5 2x10 25-161 34-161 * 28 ‐ 161 36-161 25-158 42-161 5 1x10 25-156 0 1000 2000 3000 4000 5000 6000 7000 0 2000 4000 6000 8000 0 20 40 60 80 100 120 140 Rate of Decrement in Fluorescence Rate of Decrement in Fluorescence Time (sec) % of GSN levels in mice injected with rGSN Control 100 120 48hours Percent Survival 100 PBS 160 % of Plasma Gelsolin Levels (pGSN) pGSN (Based on Western Blot) 80 75 140 GSN 60 120 40 G1-G3 20 100 50 28-161 0 80 120 # ## G4-G6 24 hours 100 60 25 80 G2-G6 40 60 20 40 0 0 Placebo GSN 0.5 mg GSN 1 mg GSN 2 mg GSN 4 mg GSN 8mg 20 Control 0 0 1 2 3 4 5 6 7 GSN + rGSN 28-161 Control G1-G3 GSN PBS GSN Days 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
Is the role of SAXS over? 120 120 EGTA 120 pH 7 pH 5 1mM Ca2+ pH 7 ∆ F [Normalized to F-actin alone] pH 6 ∆ F [Normalized to F-actin alone] ∆ F [Normalized to F-actin alone] 80 100 100 pH 5 40 80 80 60 0 60 120 1mM EGTA 1mM Ca 2+ 40 40 80 20 20 40 0 0 0 6 N 3 2 1 1 1 1 N 2 1 1 1 6 N N 3 1 G S G G 6 6 6 6 S G G G 6 6 6 6 1 1 1 1 S S - G G - - 2 1 1 - - - - - - 1 1 1 1 6 N 1 1 1 1 1 8 6 5 2 G G - G G G 1 6 2 1 1 - - - - G 6 6 6 6 6 5 5 S T 2 3 4 1 5 6 2 1 1 1 G G G - 1 1 1 1 d T 2 3 4 G 2 - - - - - - - d 5 8 0 2 4 8 5 G 2 2 3 3 3 2 2 ∆ T and G1 ‐ G3 are Ca 2+ /pH independent but when we chop further….? 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 of the glomerulus, which in turn are formed by specialized cells called podocytes. Podocytes have interdigitated shape known as foot processes. Foot process FAT Actin Cadherin Interaction between Nephrin Cytoplasmic domain of Neph1 and PDZ1 Neph1 domain of ZO ‐ 1 is ZO-1 somehow critical Podocin Membrane for functional shape of 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. Indirect Fourier transformation Concentration Unliganded Molecular proteins mass D max R g I 0 mg/ml μ m Å Å kDa Hen egg white 44 14.2 ± 0.01 14 14.2 1 a lysozyme His ‐ ZO ‐ 1 ‐ PDZ1 12.1 50 15.6 ± 0.01 66 9.5 460 Sample 1 50 15.7 ± 0.01 49 7 339 Sample 2 50 15.6 ± 0.02 24 3.4 165 Sample 3 50 15.7 ± 0.07 12 1.7 82 Sample 4 His ‐ Neph1 ‐ CD 35 70 21.3 ± 0.03 152 4.4 125 Sample 1 70 21.4 ± 0.05 93 2.7 77 Sample 2 I 0 is defined as the intensity of 70 21.4 ± 0.07 41 1.2 34 Sample 3 scattering at zero angles, is directly proportional to the Neph1 ‐ CD 22 70 18.2 ± 0.3 11 0.5 22 product of molar concentration Sample 1 and the molecular mass of the 70 18.3 ± 0.6 6.5 0.3 13.6 Sample 2 scattering sample and can be GST ‐ Neph1 ‐ 53 CD approximated by extrapolating the 110 24.1 ± 0.2 15.7 0.3 5.6 SAXS data to Q ∼ 0. Sample 1 Mallik et al 2012
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 Percentag Molar ratio of Expected I 0 e of 1:1 His ‐ ZO ‐ 1/His ‐ Neph1 D max R g I 0 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 The KIN1 motif highlighted in yellow. significance Helmstädter et al 2012 Arif et al Script being composed
Now, coming back to original problem Docking Score Few molecules Filters 1:1 Complex 1:1 Complex 2 Hours 2 Hours 14 Hours 14 Hours Neph1-CD Absorbance @280nm PDZ1 ZO-1 24 Hours Neph1-CD Absorbance @280nm PDZ1 ZO-1 24 Hours 48 Hours 48 Hours 32 34 36 38 40 32 34 36 38 40 Elution Time (mL) Elution Time (mL) 1 0 0 1 0 0 Percentage of peak area under complex 2 h o u rs 8 0 Percentage of peak area under complex 1 4 h o u rs 2 h ou rs vs. all peaks in FPLC profiles NO BINDING OCCURS BETWEEN 2 4 h o u rs 14 ho urs 8 0 vs. all peaks in FPLC profiles 24 ho urs 4 8 h o u rs ∆ THV Neph1-CD/ZO-1-PDZ1 6 0 6 0 h o u rs 48 ho urs 60 ho urs 6 0 4 0 4 0 2 0 0 2 0 N O ID 50 0 0 50 0 5 0 5 50 0 0 50 0 5 0 1 0 5 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 ) 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 )
Supplementing GFC data Keeping Fingers crossed Provisional coverage Filed
Hour-Glass Model of the Flu Infectivity Crazy Example
Shape of HA trimer 1 .0 pH 8 100 pH 7.5 pH 6.7 Normalized Log I 0 pH 5.7 pH 4.7 Log 10 I(Q) 10 pH 3 1 0.1 0 .5 0.01 0.1 0 .0 0 0 2 0 .0 0 0 4 0 .0 0 0 6 Log 10 Q 2 Q
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 Maximum Linear Dimension (Å) Maximum Linear Dimension (Å) Maximum Linear Dimension (Å) Maximum Linear Dimension (Å) Maximum Linear Dimension (Å) Native HA Native HA Native HA Native HA Native HA +Peptide P1 +Peptide P1 +Peptide P1 +Peptide P1 +Peptide P1 600 600 600 600 600 +Peptide P2 +Peptide P2 +Peptide P2 +Peptide P2 +Peptide P2 +Peptide P3 +Peptide P3 +Peptide P3 +Peptide P3 +Peptide P3 +Peptide P4 +Peptide P4 +Peptide P4 +Peptide P4 +Peptide P4 500 500 500 500 500 +Peptide P5 +Peptide P5 +Peptide P5 +Peptide P5 +Peptide P5 400 400 400 400 400 300 300 300 300 300 200 200 200 200 200 100 100 100 100 100 8 7 6 5 4 3 8 7 6 5 4 3 8 7 6 5 4 3 8 7 6 5 4 3 8 7 6 5 4 3 pH pH pH pH pH H1N1 H5N1 H9N2 H3N2 H14N5 Patent Filed
Test Case
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