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PDBsum entry 2nyy
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protein metals Protein-protein interface(s) links
Toxin/immune system PDB id
2nyy

 

 

 

 

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Contents
Protein chains
1267 a.a. *
216 a.a. *
217 a.a. *
Metals
_CA
_ZN
* Residue conservation analysis
PDB id:
2nyy
Name: Toxin/immune system
Title: Crystal structure of botulinum neurotoxin type a complexed with monoclonal antibody cr1
Structure: Botulinum neurotoxin type a. Chain: a. Cr1 monoclonal antibody. Chain: c. Fragment: light chain. Engineered: yes. Cr1 monoclonal antibody. Chain: d. Fragment: heavy chain.
Source: Clostridium botulinum. Organism_taxid: 1491. Strain: type a1 (hall strain). Homo sapiens. Human. Organism_taxid: 9606. Expressed in: cricetulus griseus. Expression_system_taxid: 10029. Expression_system_cell_line: cho dg44 cells.
Resolution:
2.61Å     R-factor:   0.208     R-free:   0.246
Authors: R.C.Stevens,J.W.Arndt
Key ref:
C.Garcia-Rodriguez et al. (2007). Molecular evolution of antibody cross-reactivity for two subtypes of type A botulinum neurotoxin. Nat Biotechnol, 25, 107-116. PubMed id: 17173035 DOI: 10.1038/nbt1269
Date:
21-Nov-06     Release date:   26-Dec-06    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
P0DPI1  (BXA1_CLOBH) -  Botulinum neurotoxin type A from Clostridium botulinum (strain Hall / ATCC 3502 / NCTC 13319 / Type A)
Seq:
Struc: 		 
Seq:
Struc: 		 
Seq:
Struc: 		1296 a.a.
1267 a.a.*
Protein chain
No UniProt id for this chain
Struc: 216 a.a.
Protein chain
No UniProt id for this chain
Struc: 217 a.a.
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 1 residue position (black cross)

 Enzyme reactions 
   Enzyme class: Chain A: E.C.3.4.24.69  - bontoxilysin.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Limited hydrolysis of proteins of the neuroexocytosis apparatus, synaptobrevins, SNAP25 or syntaxin. No detected action on small molecule substrates.
      Cofactor: Zn(2+)

 

 
DOI no: 10.1038/nbt1269 Nat Biotechnol 25:107-116 (2007)
PubMed id: 17173035  
 
 
Molecular evolution of antibody cross-reactivity for two subtypes of type A botulinum neurotoxin.
C.Garcia-Rodriguez, R.Levy, J.W.Arndt, C.M.Forsyth, A.Razai, J.Lou, I.Geren, R.C.Stevens, J.D.Marks.
 
  ABSTRACT  
 
Broadening antibody specificity without compromising affinity should facilitate detection and neutralization of toxin and viral subtypes. We used yeast display and a co-selection strategy to increase cross-reactivity of a single chain (sc) Fv antibody to botulinum neurotoxin type A (BoNT/A). Starting with a scFv that binds the BoNT/A1 subtype with high affinity (136 pM) and the BoNT/A2 subtype with low affinity (109 nM), we increased its affinity for BoNT/A2 1,250-fold, to 87 pM, while maintaining high-affinity binding to BoNT/A1 (115 pM). To find the molecular basis for improved cross-reactivity, we determined the X-ray co-crystal structures of wild-type and cross-reactive antibodies complexed to BoNT/A1 at resolutions up to 2.6 A, and measured the thermodynamic contribution of BoNT/A1 and A2 amino acids to wild-type and cross-reactive antibody binding. The results show how an antibody can be engineered to bind two different antigens despite structural differences in the antigen-antibody interface and may provide a general strategy for tuning antibody specificity and cross-reactivity.
 
  Selected figure(s)  
 
Figure 2.
Figure 2. Overview and specific interactions of the CR1-BoNT/A1 co-crystal. (a) Overall view of BoNT/A1 (yellow) in complex with the CR1 Fab with its light and heavy chains in magenta and green, respectively. (b) Overview of the CR1-BoNT/A1 interface, with the antigen contacting loops (H1, H2, H3, L1, L2 and L3) and toxin -strands indicated. (c) Detailed view of contacts between CR1 Fab and BoNT/A1. A cartoon representation of BoNT/A1 is shown with carbons (yellow), nitrogens (blue) and oxygens (red). Amino acid contacts are indicated by magenta (V[L]), green (V[H]) and black (BoNT/A) numbering. V[L], variable light chain; V[H], variable heavy chain. 		Figure 3.
Figure 3. Location of interactions that differ between CR1 and BoNT/A1 and BoNT/A2 and affect differential BoNT/A binding. (a) Location of CR1-BoNT/A1 contact residues and residues differing between BoNT/A1 and BoNT/A2. The alignment of BoNT/A1 and A2 subtypes shows strict sequence conservation in white letters on red background, and strong sequence conservation in red letters. The residues composing the CR1 epitope of the H[CN] lectin (residues 874–1094) and H[CC] trefoil (residues 1095–1295) subdomains are indicated with red triangles, energetically important residues are shown with black triangles. Disulfide bonds are indicated using green numbers. The secondary structure elements of the BoNT/A1 binding domain structure are labeled ( -helix), ( -strand) and TT (turn). (b) Structural location of differences between BoNT/A1 and A2 and impact on CR1 interactions. (i,ii) Surface representations of BoNT/A1 (yellow) in complex with CR1 (V[L] in magenta and V[H] in green) showing patches of sequence variability between BoNT/A1 and BoNT/A2 subtypes in slate blue. (iii,iv) Close-up view of sequence variability between T1063 and H0164 of BoNT/A1 (yellow, iii) and modeled P1063 and R0164 of BoNT/A2 (cyan, iv) in complex with CR1 (V[L] in magenta and V[H] in green). (v,vi) Surface representations of BoNT/A1 (yellow) with BoNT/A1 and BoNT/A2 sequence differences in slate blue. Key differences between BoNT/A1 and BoNT/A2 that are functionally important for binding (high G values) are shown in dark blue (1063 and 1064). Functionally important BoNT/A residues (high G values) that do not differ between BoNT/A1 and BoNT/A2 are shown in red. Panel v shows CR1 with its light and heavy chains in magenta and green, respectively, with its H1 loop in tan. Panel vi is looking down onto the CR1 epitope of BoNT/A1 with CR1 removed. (c) Details of the interaction between AR2 and CR1 and BoNT/A1 and A2 at the H1 loop. Close-up view of the H1 loop showing sequence and structural differences between BoNT/A1 (yellow) and BoNT/A2 (cyan), in complex with the CR1 (green) and AR2 (orange) Fabs. The BoNT/A2-CR1 and BoNT/A2-AR2 structures are modeled.
 
  The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Biotechnol (2007, 25, 107-116) copyright 2007.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21390033 A.R.Bradbury, S.Sidhu, S.Dübel, and J.McCafferty (2011).
Beyond natural antibodies: the power of in vitro display technologies.
  Nat Biotechnol, 29, 245-254.  
21149386 C.Garcia-Rodriguez, I.N.Geren, J.Lou, F.Conrad, C.Forsyth, W.Wen, S.Chakraborti, H.Zao, G.Manzanarez, T.J.Smith, J.Brown, W.H.Tepp, N.Liu, S.Wijesuriya, M.T.Tomic, E.A.Johnson, L.A.Smith, and J.D.Marks (2011).
Neutralizing human monoclonal antibodies binding multiple serotypes of botulinum neurotoxin.
  Protein Eng Des Sel, 24, 321-331.  
20410329 D.M.Held, A.C.Shurtleff, S.Fields, C.Green, J.Fong, R.G.Jones, D.Sesardic, R.Buelow, and R.L.Burke (2010).
Vaccination of rabbits with an alkylated toxoid rapidly elicits potent neutralizing antibodies against botulinum neurotoxin serotype B.
  Clin Vaccine Immunol, 17, 930-936.  
20123883 H.Iwai, B.Oztürk, M.Ihara, and H.Ueda (2010).
Antibody affinity maturation in vitro using unconjugated peptide antigen.
  Protein Eng Des Sel, 23, 185-193.  
20138889 J.Dong, A.A.Thompson, Y.Fan, J.Lou, F.Conrad, M.Ho, M.Pires-Alves, B.A.Wilson, R.C.Stevens, and J.D.Marks (2010).
A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic alpha-exosite binding region.
  J Mol Biol, 397, 1106-1118.
PDB code: 3k3q
20156888 J.Lou, I.Geren, C.Garcia-Rodriguez, C.M.Forsyth, W.Wen, K.Knopp, J.Brown, T.Smith, L.A.Smith, and J.D.Marks (2010).
Affinity maturation of human botulinum neurotoxin antibodies by light chain shuffling via yeast mating.
  Protein Eng Des Sel, 23, 311-319.  
20098614 J.O.Conway, L.J.Sherwood, M.T.Collazo, J.A.Garza, and A.Hayhurst (2010).
Llama single domain antibodies specific for the 7 botulinum neurotoxin serotypes as heptaplex immunoreagents.
  PLoS One, 5, e8818.  
19917718 J.Sepulveda, J.Mukherjee, S.Tzipori, L.L.Simpson, and C.B.Shoemaker (2010).
Efficient serum clearance of botulinum neurotoxin achieved using a pool of small antitoxin binding agents.
  Infect Immun, 78, 756-763.  
20233039 M.Montal (2010).
Botulinum neurotoxin: a marvel of protein design.
  Annu Rev Biochem, 79, 591-617.  
19903738 S.A.Kenrick, and P.S.Daugherty (2010).
Bacterial display enables efficient and quantitative peptide affinity maturation.
  Protein Eng Des Sel, 23, 9.  
20808925 S.R.Kalb, C.Garcia-Rodriguez, J.Lou, J.Baudys, T.J.Smith, J.D.Marks, L.A.Smith, J.L.Pirkle, and J.R.Barr (2010).
Extraction of BoNT/A, /B, /E, and /F with a single, high affinity monoclonal antibody for detection of botulinum neurotoxin by Endopep-MS.
  PLoS One, 5, e12237.  
19477127 C.J.Farady, B.D.Sellers, M.P.Jacobson, and C.S.Craik (2009).
Improving the species cross-reactivity of an antibody using computational design.
  Bioorg Med Chem Lett, 19, 3744-3747.  
  19149692 J.C.Pai, J.N.Sutherland, and J.A.Maynard (2009).
Progress towards recombinant anti-infective antibodies.
  Recent Pat Antiinfect Drug Discov, 4, 1.  
19054791 J.Gabbard, N.Velappan, R.Di Niro, J.Schmidt, C.A.Jones, S.M.Tompkins, and A.R.Bradbury (2009).
A humanized anti-M2 scFv shows protective in vitro activity against influenza.
  Protein Eng Des Sel, 22, 189-198.  
19256469 J.S.Henkel, M.Jacobson, W.Tepp, C.Pier, E.A.Johnson, and J.T.Barbieri (2009).
Catalytic properties of botulinum neurotoxin subtypes A3 and A4.
  Biochemistry, 48, 2522-2528.  
19381395 J.W.Grate, M.G.Warner, R.M.Ozanich, K.D.Miller, H.A.Colburn, B.Dockendorff, K.C.Antolick, N.C.Anheier, M.A.Lind, J.Lou, J.D.Marks, and C.J.Bruckner-Lea (2009).
Renewable surface fluorescence sandwich immunoassay biosensor for rapid sensitive botulinum toxin detection in an automated fluidic format.
  Analyst, 134, 987-996.  
19651864 L.W.Cheng, L.H.Stanker, T.D.Henderson, J.Lou, and J.D.Marks (2009).
Antibody protection against botulinum neurotoxin intoxication in mice.
  Infect Immun, 77, 4305-4313.  
19643593 M.G.Warner, J.W.Grate, A.Tyler, R.M.Ozanich, K.D.Miller, J.Lou, J.D.Marks, and C.J.Bruckner-Lea (2009).
Quantum dot immunoassays in renewable surface column and 96-well plate formats for the fluorescence detection of botulinum neurotoxin using high-affinity antibodies.
  Biosens Bioelectron, 25, 179-184.  
19111565 M.Montal (2009).
Translocation of botulinum neurotoxin light chain protease by the heavy chain protein-conducting channel.
  Toxicon, 54, 565-569.  
  20069756 S.Fagète, U.Ravn, F.Gueneau, G.Magistrelli, M.H.Kosco-Vilbois, and N.Fischer (2009).
Specificity tuning of antibody fragments to neutralize two human chemokines with a single agent.
  MAbs, 1, 288-296.  
19399171 S.R.Kalb, J.Lou, C.Garcia-Rodriguez, I.N.Geren, T.J.Smith, H.Moura, J.D.Marks, L.A.Smith, J.L.Pirkle, and J.R.Barr (2009).
Extraction and inhibition of enzymatic activity of botulinum neurotoxins/A1, /A2, and /A3 by a panel of monoclonal anti-BoNT/A antibodies.
  PLoS ONE, 4, e5355.  
19610073 S.S.Hall, and P.S.Daugherty (2009).
Quantitative specificity-based display library screening identifies determinants of antibody-epitope binding specificity.
  Protein Sci, 18, 1926-1934.  
18947189 E.R.Goldman, G.P.Anderson, J.Conway, L.J.Sherwood, M.Fech, B.Vo, J.L.Liu, and A.Hayhurst (2008).
Thermostable llama single domain antibodies for detection of botulinum A neurotoxin complex.
  Anal Chem, 80, 8583-8591.  
18446228 K.Bagramyan, J.R.Barash, S.S.Arnon, and M.Kalkum (2008).
Attomolar detection of botulinum toxin type A in complex biological matrices.
  PLoS ONE, 3, e2041.  
18656541 L.G.Presta (2008).
Molecular engineering and design of therapeutic antibodies.
  Curr Opin Immunol, 20, 460-470.  
18251694 L.M.Zarebski, K.Vaughan, J.Sidney, B.Peters, H.Grey, K.D.Janda, A.Casadevall, and A.Sette (2008).
Analysis of epitope information related to Bacillus anthracis and Clostridium botulinum.
  Expert Rev Vaccines, 7, 55-74.  
18336206 L.R.Pepper, Y.K.Cho, E.T.Boder, and E.V.Shusta (2008).
A decade of yeast surface display technology: where are we now?
  Comb Chem High Throughput Screen, 11, 127-134.  
18704164 P.Stenmark, J.Dupuy, A.Imamura, M.Kiso, and R.C.Stevens (2008).
Crystal structure of botulinum neurotoxin type A in complex with the cell surface co-receptor GT1b-insight into the toxin-neuron interaction.
  PLoS Pathog, 4, e1000129.
PDB codes: 2vu9 2vua
18237741 S.M.Lynch, C.Zhou, and A.Messer (2008).
An scFv intrabody against the nonamyloid component of alpha-synuclein reduces intracellular aggregation and toxicity.
  J Mol Biol, 377, 136-147.  
17676054 A.J.Link, K.J.Jeong, and G.Georgiou (2007).
Beyond toothpicks: new methods for isolating mutant bacteria.
  Nat Rev Microbiol, 5, 680-688.  
17211401 L.Presta (2007).
Evolving an anti-toxin antibody.
  Nat Biotechnol, 25, 63-65.  
17716905 N.Congy-Jolivet, A.Probst, H.Watier, and G.Thibault (2007).
Recombinant therapeutic monoclonal antibodies: mechanisms of action in relation to structural and functional duality.
  Crit Rev Oncol Hematol, 64, 226-233.  
17418361 R.Pantophlet, R.O.Aguilar-Sino, T.Wrin, L.A.Cavacini, and D.R.Burton (2007).
Analysis of the neutralization breadth of the anti-V3 antibody F425-B4e8 and re-assessment of its epitope fine specificity by scanning mutagenesis.
  Virology, 364, 441-453.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB code is shown on the right.

 

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