Fast Association Rates Suggest a Conformational Change in the MHC Class I Molecule H-2D^bupon Peptide Binding

Abstract

Major histocompatibility complex (MHC) class I molecules bind peptides in the endoplasmic reticulum (ER). For this binding reaction, when performed in vitro, widely differing association rates have been reported. We have expressed empty soluble H-2Db class I molecules in Chinese hamster ovary (CHO) cells and generated complete sets of association, dissociation, and equilibrium constants of unmodified peptides using tritium-labeled peptides and stopped-flow fluorescence spectroscopy. We find that (i) the transition midpoint of temperature denaturation (Tm) of the protein is shifted from 30.5 to 56 degrees C upon the binding of a high-affinity peptide. (ii) With the peptide SV-324-332 (sequence FAPGNYPAL) at 4 degrees C, the dissociation rate constant of 1.02 x 10(-5) s-1 and an equilibrium constant of 8.5 x 10(7) M-1 predict an association rate constant of 870 M-1 s-1 for a simple one-step model of binding. (iii) In contrast, binding of this peptide proceeds much faster, with 1.4 x 10(6) M-1 s-1. These "mismatch kinetics" suggest that peptide binding occurs in several steps, most likely via a conformational rearrangement of the peptide binding groove. The structure of the peptide-class I complex at the time-point of peptide recognition may therefore be different from the equilibrium crystal structures. (iv) Association of modified peptides, in the presence of detergent, or above the Tm of the empty molecule is considerably slower. This might explain why fast on-rates have not been observed in previous studies.

Promiscuous binding of extracellular peptides to cell surface class I MHC protein

“…When expressed in conventional units (M −1 s −1 ) rather than in the units (M −1 h −1 ) used in Table 1, we found 4,900 M −1 s −1 for OVA-K b and 217 M −1 s −1 for SIY-K b (Table 1). In contrast, the highest k on reported was about 10 6 M −1 s −1 for a nonamer binding to D b (33). The latter, however, is still 10-100 times slower than the value found for some antibody-hapten reactions; for instance, the k on for binding an ε-2,4-DNP-lysine nonamer [DNP-(lysine) 9 ] by a homogeneous antibody (myeloma protein) was 10 7 M −1 s −1 , and the same protein's binding of the smaller hapten, ε-2,4-DNP-lysine, was 10 8 M −1 s −1 , which was almost diffusion limited (SI Text, R18 and R19).…”

“…A basis for these disparate k on values is suggested by studies in which the binding of dansyl-or fluorescein-labeled peptides to recombinant MHC proteins was monitored continuously (13)(14)(15)33). Under these conditions, the association kinetics were biphasic: one rate, reflecting a slow unimolecular step, was attributed to changes of MHC conformation from peptide-unreceptive to -receptive conformations, and the other rate was attributed to peptide binding to the MHC's receptive conformation.…”

Tapasin edits peptides on MHC class I molecules by accelerating peptide exchange

“…The corresponding association rate, k 1 , should be in the same order of magnitude; if the peptide concentration inside the membranes is identical to that outside, it is at least 5000 M À1 s À1 , but it could be much larger. We have previously measured 35 000 in detergent lysates, and 2 Â 10 6 with recombinant protein [33]. The rate of conversion of the unstable CP Ã form to the stable form (CP) is about 0.001 s À1 for the WT K b molecule in the presence of tapasin, and about 0.01 s À1 for T134K (since in Fig.…”

“…(2) explains why in the literature, two ranges of association constants for peptide onto class I have been found, depending on the method used to investigate them. With real-time stopped-flow label-free tryptophan fluorescence spectroscopy, which does not separate bound from free ligand, association rates in the range of 2 Â 10 6 M À1 s À1 were observed [33], while immunoprecipitation methods, which test the establishment of a biochemically stable complex with a slow off-rate, found only 10 4 M À1 s À1 [34]. Most likely, the former data represent the fast formation of an encounter complex, while the latter show the slow conversion to the stable form.…”

Inhibitory mechanism of peptides with a repeating hydrophobic and hydrophilic residue pattern on interleukin-10

“…To strengthen the helical conformation, P1 or P2 don't have the breaking pattern observed at Val33. It has been noted that the biophysical properties of protein-binding peptides, such as main chain hydrogen bond forming ability with target protein, sequence length, secondary structures, side-chain/main-chain, salt bridge, [25][26][27][28][29] affect its binding activity directly. In this study, we thus synthesized sequence-modified peptides (P1.1 to 1.5, P2.1 to 2.3 listed in Table 1) based on P1 and P2, to further clarify the sequential characteristics that could influence IL-10 inhibitory activity.…”