Empty Class II Major Histocompatibility Complex Created by Peptide Photolysis Establishes the Role of DM in Peptide Association

Gijsbert M Grotenbreg,Melissa J Nicholson,Kevin D Fowler,Kathrin Wilbuer,Leah Octavio,Maxine Yang,Arup K Chakraborty,Hidde L Ploegh,Kai W Wucherpfennig

doi:10.1074/jbc.m702844200

Abstract

DM catalyzes the exchange of peptides bound to Class II major histocompatibility complex (MHC) molecules. Because the dissociation and association components of the overall reaction are difficult to separate, a detailed mechanism of DM catalysis has long resisted elucidation. UV irradiation of DR molecules loaded with a photocleavable peptide (caged Class II MHC molecules) enabled synchronous and verifiable evacuation of the peptide-binding groove and tracking of early binding events in real time by fluorescence polarization. Empty DR molecules generated by photocleavage rapidly bound peptide but quickly resolved into species with substantially slower binding kinetics. DM formed a complex with empty DR molecules that bound peptide with even faster kinetics than empty DR molecules just having lost their peptide cargo. Mathematical models demonstrate that the peptide association rate of DR molecules is substantially higher in the presence of DM. We therefore unequivocally establish that DM contributes directly to peptide association through formation of a peptide-loading complex between DM and empty Class II MHC. This complex rapidly acquires a peptide analogous to the MHC class I peptide-loading complex.

Highlights

Peptide binding may at first glance appear to be a rather simple process, in which a peptide interacts with an available Class II major histocompatibility complex (MHC) site
Closer inspection reveals the presence of multiple distinct species of different behavior and stability [12, 26, 40]: (i) stable DR-peptide complexes from which peptides dissociate at very slow rates; (ii) a transition state induced by DM from which peptide dissociates rapidly; (iii) a labile empty DR conformer that rapidly binds peptide, but is short-lived; (iv) inactive and aggregated forms; and (v) DM-bound empty DR molecules that are stable and bind peptide rapidly
When invariant chain degradation was blocked by leupeptin, DM could be retrieved in complex with DR molecules together with a 21-kDa N-terminal fragment of the invariant chain

Summary

Introduction

The MBP-488 reporter peptide was included during photocleavage, and the reaction had already proceeded significantly when the first data points could be recorded, even though photocleavage was performed for 2 min on ice. in the absence of DM, rapid peptide association occurs to what we propose are newly vacated DR2 molecules. The peptide binding reaction proceeded rapidly following photocleavage in the absence of DM, but DM further accelerated the rate of peptide association (Fig. 3D).

Results

Conclusion