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Unique structural and functional features of Botulinum Neurotoxin
By
Dr. RAJ KUMAR At University of Massachusetts, Lowell
7th Nov. 2013
Toxins are poisonous substances produced by microbes including bacteria, parasites, fungi, viruses, animals, and plants that are poisonous to other species.
Exo S /c3 exotoxin
SPEB
1. Toxin acting on the cell surface, 2. AB types of toxin 3. Toxins that are delivered to the host cells by bacteria.
Botulinum Toxin : Background
--Toxin produced by the bacterium Clostridium botulinum --Anaerobic, gram positive, rod-shaped bacteria --Bacteria are 0.5 to 2.0 µm in width and 1.6 to 22.0 µm in length --Create spores that can remain dormant for 30 years or more --Spores extremely resistant to environmental stressors, such as heat and UV light.
--8 types of botulinum neurotoxins - A through H, based on the antigenic properties of the toxin produced
toxins A, B, E and F cause illness in humans
toxins C and D cause illness in birds and mammals
toxin G
– All seven serotypes are structurally same but immunologically distinct. Bioterrorism!
-LD50 is 1 nanogram per kilogram of body weight = VERY POTENT
BoNT Molecule : A generous gift from nature
BoNTs has been used in three areas of medicine:
Dermatology
Opthamology
neurology
Truong, D.; Jost, W. Parkinsonism Relat D 2006, 12, 331-355. ; Brin, M. Toxicon 2009, 54, 676-682.
Kumar et al., 2013
1. Preparation of novel vaccines 2. Delivery of therapeutic molecules by using binding domain as a transporter 3. Transporter of cargo proteins, DNA and antibodies
Translocation of therapeutic proteins to cytosol
1. Inhibition of hypersecretion 2. Retargeting LC function
BoNT Molecule : Poison or Nectar
1. Functional Uniqueness 2. Structural Uniqueness 3. Evolutionary Traits
Sections of presentation
Functional Uniqueness
BoNT/ A, B and E BoNT/ C and D BoNT/F BoNT/C
Clostridium botulinum
Neurotoxin
Singh, Kumar, and Cai, Handbook of Neurotoxicity, 2013
Binz et.al. 2009
BoNT Molecule : Unique Trimodular structure
Substarte: SNARE Proteins SNAP-25 VAMP Syntaxin
1-448 LC 492-545 Belt 548-872 HN
873-1296 HC
Endocytosis and Exocytosis Process and role of SNARE
Synaptic cleft
Steps
1. Membrane Binding
2. Internalization
3. Translocation
4. Clevage of SNARE (Endopeptidase activity)
Kumar et.al., Springer 2013 (in press)
Unique mechanism of action
Blockage of neurotransmitter release
Method of translocation
Copyright (2007) © National Academy of Science, USA.
Dhaliwal et al., (manuscript in preparation)
Surv
ival
tim
e (h
)
01224364860728496
DrBoNT/A TmDD-mutantDhaliwal et al., (manuscript in preparation)
Functional uniqueness of this molecule
Structure uniqueness of this molecule
1. Unique trimodular structure
2. Hc belt
1. Binding Specificity
Low affinity binding to ganglioside and high affinity binding to respective neuronal receptor. 2. SNARE Specificity
Specific SNARE substrate and cleavage site is very specific.
3. Duration of inhibition of neurotransmitter release
BoNT/A is persistant for 180 days BoNT/B is persistant for 90 days BoNT/E is persistant for 30 days
4. Optimally active molten globule enzyme
Brunger et.al. 2007
Active Site
1. Zn-mettalloprotease family ---- Zn-binding motif HEXXH+E.
2.α/β globular protein.
3.Recognize tertiary structure of the substrate.
4. Cleavage site is specific site out of several identical peptide bonds present in their respective target protein.
5. Two exosites : α and β.
6. Loops 50/60, 60/70, 170, 250 and 370 play major roles in recognition and activity.
7. Exceptionally high stability inside the cell. 8. Solution structure is different from published crystal structure.
9. Active molten globule.
10. Flexible active site.
Unique Properties of Botulinum Neurotoxin Endopeptidase
Brunger et.al. 2004
Conformational Uniqueness
Protein Folding energy Landscape
Onuchic et al. Annu. Rev. Phys. Chem. 1997. 48:545-600
Native Protein :
Functional form of a protein ---- unbranched chain of amino acids in a specific 3D shape.
Native structure ---- collection of whole ensemble of conformations which are populated under certain physiological condition.
Molten globule state Activity
Cai and Singh, 2001
Kukreja and Singh, 2005Pre imminent molten globule enzyme
PRIME structure of BoNT/A LC
β-exosite
Binding and recognition of substrate
Briedenbach and Brunger, 2004
Unique folding
Pink : α-helix Yellow : β-sheets Blue : 310-helix Green : turns White : unstructured
Kumar et al., JBSD, 2013
Simulation timeline
Kumar et al., JBSD, 2013
2ISE- LCA
2ISG- LCB
1T3A- LCE
Tertiary Structure
Folding
Kumar et al., JBSD, 2013
SAXS Intensity PDDF plot
25 C 37 C
Kumar et al. (manuscript in preparation)
Conformation of BoNT/A LC at 25°C and 37°C
Solution structure of BoNT endopeptidase
Active site of BoNT/A LC
Extra structure?
Aligned Structure
Is this extra structure is of c-terminus of BoNT/A LC?
Full length LCA (454 a.a) PDB structure of LCA (425 a.a)
TLCA
25°C
TLCA
LCA
37°C
Evolutionary Traits
Forms of Life • Bacteria • Archaea • Eukarya
Kumar et al., 2013, Springer (in press)
Pál et al. Nature Reviews Genetics 7, 337–348 (May 2006) | doi:10.1038/nri1838
General View of Protein Evolution
Kumar et al., 2013, Springer (in press)
+ Hemagglutinin Proteins
to interlukins, ricin like lectins, laminin, fibroblast growth factors , and perisin.
Endopeptidase Domain
Similar to
Zn-metalloprotease
HEXXH
Notable points:
1) The percent identity among BoNTs at the amino acid levels range from 34.1% to 98.2%.
2) The sequence homologies of NBPs are higher than BoNTs genes which suggests that NBPs are more conserved than BoNTs.
3) BoNTs protein sequence is unrelated to other protein sequences.
4) Protein evolution tree shows that BoNT/E is the latest toxin.
Kumar et al., 2013, Springer (in press)
Conclusions
1. Evolutionary origin of Botulinum toxin is unknown.
2. Possible co-evolution with substrate.
3. Translocation of LC is still unclear. Investigation at molecular level is going on.
4. Solution structure conformation of BoNT/A LC appears to be different compared to published crystal structure conformation. Solution conformation of BoNT/A LC is different at 37°C compared to 25°C, which can explain the existence of PRIME structure.
5. Existence of active molten and pre-molten globule conformation indicates flexibility of enzyme. This dimension must be utilize to develop an effective countermeasures.
6. Investigation of structural and functional features of this molecules can reveal some unique features of protein.
Botulinum Research Centre, UMASS, Dartmouth
Dr. Bal Ram Singh Dr. S. Cai Dr. Roshan Kukreja
UMASS, Lowell
Dr. Valeri Barsegov
Brookheaven National Laboratory
Dr. Subramanian Swaminathan Dr. Desigan Kumaran
NMRFAM, University of Wisconsin
Dr. Jordon Burke Dr. Milo Westler
Funding Agencies
USAMRID, DOD, NIH