PMID- 10998256
OWN - NLM
STAT- MEDLINE
DCOM- 20001013
LR  - 20190613
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 39
IP  - 36
DP  - 2000 Sep 12
TI  - Assembly and dissociation of human leukocyte antigen (HLA)-A2 studied by 
      real-time fluorescence resonance energy transfer.
PG  - 11163-9
AB  - Class I major histocompatibility complex (MHC) heterodimer, composed of human 
      leukocyte antigen (HLA)-A2 heavy chain and human beta(2)-microglobulin 
      (beta(2)m), was produced by denaturation and gel filtration of the recombinant 
      water-soluble HLA-A2/beta(2)m/peptide ternary complex in 8 M urea Tris-HCl 
      buffer, followed by refolding of the separated chains without peptide. Peptide 
      affinity and kinetics of the ternary complex formation and dissociation were 
      investigated in real time by monitoring the fluorescence resonance energy 
      transfer (FRET) from intrinsic HLA-A2 heavy-chain tryptophans to a dansyl 
      fluorophore conjugated to the bound peptide. Peptide binding to the heterodimer 
      was a second order process with rate constants linearly dependent upon 
      temperature in Arrhenius coordinates over 0-20 degrees C. The binding rate 
      constant of pRT6C-dansyl [ILKEPC(dansyl)HGV] at 37 degrees C evaluated by 
      extrapolation of the Arrhenius plot was (2.0 +/- 0.5) x 10(6) M(-1) s(-1). 
      Association of the heavy chain with beta(2)m was a first order process, 
      apparently controlled by a conformational transition in the heavy chain. One of 
      these conformations bound to beta(2)m to form the heavy chain/beta(2)m 
      heterodimer whereas the second conformer oligomerized. Peptide dissociation from 
      the ternary complex was a first-order reaction over the temperature range 20-37 
      degrees C, suggesting that the ternary complex also exists in two conformations. 
      Taken together, the present data suggest that association of beta(2)m changes the 
      HLA-A2 heavy-chain conformation thereby promoting peptide binding. Peptide 
      dissociation from the ternary complex induces dissociation of the 
      heavy-chain/beta(2)m heterodimer thereby causing oligomerization of the heavy 
      chain. The lability of the HLA-A2/beta(2)m heterodimer and the strong tendency of 
      the "free" heavy chain to oligomerize may provide an efficient mechanism for 
      control of antigen presentation under physiological conditions by reducing the 
      direct loading of HLA with exogenous peptide at the cell surface.
FAU - Gakamsky, D M
AU  - Gakamsky DM
AD  - Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, 
      Israel. lidima@wis.weizmann.ac.il
FAU - Davis, D M
AU  - Davis DM
FAU - Strominger, J L
AU  - Strominger JL
FAU - Pecht, I
AU  - Pecht I
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (HLA-A2 Antigen)
RN  - 0 (Peptides)
RN  - 0 (beta 2-Microglobulin)
SB  - IM
MH  - Dimerization
MH  - *Energy Transfer
MH  - HLA-A2 Antigen/*chemistry/*metabolism
MH  - Humans
MH  - Kinetics
MH  - Peptides/chemistry/immunology/metabolism
MH  - Protein Binding/immunology
MH  - Protein Conformation
MH  - Protein Processing, Post-Translational/immunology
MH  - Spectrometry, Fluorescence/methods
MH  - Thermodynamics
MH  - Titrimetry
MH  - beta 2-Microglobulin/chemistry/metabolism
EDAT- 2000/09/22 11:00
MHDA- 2000/10/21 11:01
CRDT- 2000/09/22 11:00
PHST- 2000/09/22 11:00 [pubmed]
PHST- 2000/10/21 11:01 [medline]
PHST- 2000/09/22 11:00 [entrez]
AID - bi000763z [pii]
AID - 10.1021/bi000763z [doi]
PST - ppublish
SO  - Biochemistry. 2000 Sep 12;39(36):11163-9. doi: 10.1021/bi000763z.