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