LIR1 interacts with classical and nonclassical MHC class 1

1
LIR-1 interacts with classical and non-classical
MHC class 1

• Joseph James 780690
• Ying Wang 759713
• Monika Jankute 778129
• Pardeep Singh Jagpal 776063

2
HLA-A2

• Human Leukocyte Antigen
• Found on the surface of a variety of somatic
  cells
• Classical Class 1 Major Histocompatability
  Complex (MHC)
• Membrane glycoprotein
• Can interact with a large number of antigens.
• Presents antigens to the T-cells via T-cell
  receptors (TCR) and the LIR

3
Leukocyte Immunoglobulin-like Receptor (LIR-1)

• Expressed on many Lymphoid and Myeloid cells
• NK cells, Monocytes, Dendrites, B cells, T cells
• Type 1 Transmembrane protein
• 4 extracellular Ig-like domains(D1-D4)
• Binds broads range of MHC class 1 molecules
• Is the receptor for Human Cytomegalovirus (HCMV)
  UL18, an MHC class 1 homologue.
• Transmits inhibitory signals through
  tyrosine-based inhibitory motifs.
• Thought to prevent cell lysis

4

MHC class I and leukocyte immunoglobulin like
receptor 1 LIR-1

• Immunoglobulin domain 1 and 2 of LIR-1
• arranged at a nearly perpendicular angle.

• Short cytoplasmic tail
• Single transmembrane helix
• Alpha-3 domain
• Alpha1 and alpha2 domain
• Beta1 microglobulin

5
Interaction between HLA-A2 and LIR-1

• The 10-16º change in the D1D2 interdomain angle
  was shown in the LIR-1-HLA-A2 complex

• Two distinct contact area
• a3 domain contacts at the end of the strand A and
  in the strand A-B loop (a.a 193-200) and the E
  strand (a.a 248).
• ß2M contacts the N-terminus (a.a 1-4), in the F-G
  strand loop (a.a 86-89) and in the G strand
  itself (a.a 91-94, 96 and 99).

6
Conserved residues in a3 domain

• In all MHC class I molecules examined
• residues Glu198 and Val248 are conserved,
  residue 193 is either a Pro or Ala and residue
  194

• is uncharged and hydrophobic (all a3 domain
  residues).
• a3 domain 195-197 are conserved in HLA-A,
  HLA-B, HLA-C, HLA-E and HLA-F.
• FcRn, HFE and ZAG have 4 to 6 a.a changes, of
  which 3 to 4 are non-conservative.
• LIR-1 recognizes a sequence motif on the a3
  domain that is restricted to classical and
  non-classical MHC class I molecules, a3 domain
  contact provides essential energetic
  contributions to binding energy.

7
Conserved residues in LIR-1

• Four (Arg36, Tyr38, Arg39 and Lys41) of the six
  LIR-1 amino acids that contact the HLA a3 domain
  are conserved in LIR-2.
• Alternative uncharged polar amino acids are
  substituted at positions 43 and 76.

8
Methods

• Recombinant Protein Expression
• Hanging Drop Precipitation
• Size Exclusion Chromatography
• Various computer modelling programs e.g.
• DENZO
• AmoRe
• CONTACT
• Molecular replacement

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• BUT..
• Only a static picture
• No real time reflection or moeity
• Ideally want to go forth and investigate binding
• particularly which residues are important e.g. D1
  Distal Tip.
• which conserved residues are key e.g. the
  conserved interacting residues of the ß2M domain

10
Further Experiments

• Binding assays of wt compared to site specific
  mutagenesis of those specific residues will give
  detail of binding affinities as well as
  identifying key residues
• Recobinant gene purification and expression in
  E.coli
• Sonicate Purify
• Ultracentrifugation incubation with substrate
• Link to surface plasmon resonance as a simple
  concept to investigase binding properties

11
Discussion

• Differences in the orientations of the ß2m and a3
  domain, though mostly conserved in MHC class1s,
  could affect strength of LIR-1 binding.
• UL18 binds LIR-1 around 3000 fold stronger than
  it does to LIR-2. This may be due in part to the
  differences in five residues on the UL18s
  binding site for LIR-1 resulting to a more
  optimum orientation of the D1D2 domains.

Adapted from Chapman, (1999)