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Overexpression, crystallization and preliminary x-ray crystallographic analysis of the variable lymphocyte receptor 2913 ectodomain fused with internalin b

Academic Article
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Overview

related to degree

  • Kirchdoerfer, Robert, Ph.D. in Molecular Biophysics, Scripps Research 2006 - 2012

authors

  • Lee, J. Y.
  • Kim, H. S.
  • Baek, I. W.
  • Back, J. M.
  • Han, M. R.
  • Kong, S. Y.
  • Kim, J. H.
  • Kirchdoerfer, Robert
  • Kim, J. O.
  • Cooper, M. D.
  • Wilson, Ian
  • Kim, H. J.
  • Han, B. W.

publication date

  • January 2013

journal

  • Acta Crystallographica Section F-Structural Biology and Crystallization Communications  Journal

abstract

  • In jawless vertebrates, variable lymphocyte receptors (VLRs) play a crucial role in the recognition of antigens as part of the adaptive immune system. Leucine-rich repeat (LRR) modules and the highly variable insert (HVI) of VLRs contribute to the specificity and diversity of antigen recognition. VLR2913, the antigen of which is not known, contains the same HVI amino-acid sequence as that of VLR RBC36, which recognizes the H-trisaccharide from human blood type O erythrocytes. Since the HVI sequence is rarely identical among all known VLRs, identification of the antigen for VLR2913 and the main contributing factors for antigen recognition based on a comparison of VLR2913 and VLR RBC36 has been attempted. To initiate and facilitate this structural approach, the ectodomain of VLR2913 was fused with the N-terminal domain of internalin B (InlB-VLR2913-ECD). Three amino-acid residues on the concave surface of the LRR modules of InlB-VLR2913-ECD were mutated, considering important residues for hydrogen bonds in the recognition of H-trisaccharide by VLR RBC36. InlB-VLR2913-ECD was overexpressed in Escherichia coli and was crystallized at 295 K using the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to 2.04 Å resolution and could be indexed in the tetragonal space group P4(1)2(1)2 (or P4(3)2(1)2), with unit-cell parameters a = 91.12, b = 91.12, c = 62.87 Å. Assuming that one monomer molecule was present in the crystallographic asymmetric unit, the calculated Matthews coefficient (V(M)) was 2.75 Å(3) Da(-1) and the solvent content was 55.2%. Structural determination of InlB-VLR2913-ECD by molecular replacement is in progress.

subject areas

  • Animals
  • Bacterial Proteins
  • Crystallography, X-Ray
  • Escherichia coli
  • Hydrogen Bonding
  • Lymphocytes
  • Membrane Proteins
  • Mutation
  • Protein Conformation
  • Protein Structure, Tertiary
  • Proteins
  • Receptors, Immunologic
  • Recombinant Proteins
  • Trisaccharides
  • Vertebrates
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Identity

PubMed Central ID

  • PMC3539700

International Standard Serial Number (ISSN)

  • 1744-3091

Digital Object Identifier (DOI)

  • 10.1107/s1744309112045484

PubMed ID

  • 23295483
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Additional Document Info

start page

  • 39

end page

  • 41

volume

  • 69

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