Gundo Diedrich

The nature of the MHC class I peptide loading complex

Summary: Peptide binding to major histocompatibility complex (MHC) dass I molecules occurs in the endoplasmic reticulum (ER). Efficient peptide binding requires a number of components in addition co the MHC class I‐β2 microglobulin dimer (β2m). These include the two subunits of the transporter associated with antigen presentation (TAP1 and TAP2), which are essential for introducing peptides into the ER from the cytosol, and tapasin, an MHC‐encoded membrane protein. Prior to peptide binding, MHC class I‐β2m dimers form part of a large multisubnnit ER complex which includes TAP and tapasin. In addition to these specialized components two soluble ‘house‐keeping’ proteins, the chaperone calreticulin and the thiol oxidoreductase ERp57, are also components of this complex. Our current understanding of the nature and function of the MHC class I peptide loading complex is the topic of this review.

A Role for Calnexin in the Assembly of the MHC Class I Loading Complex in the Endoplasmic Reticulum

Heterodimers of MHC class I glycoprotein and β2-microglobulin (β2m) bind short peptides in the endoplasmic reticulum (ER). Before peptide binding these molecules form part of a multisubunit loading complex that also contains the two subunits of the TAP, the transmembrane glycoprotein tapasin, the soluble chaperone calreticulin, and the thiol oxidoreductase ERp57. We have investigated the assembly of the loading complex and provide evidence that after TAP and tapasin associate with each other, the transmembrane chaperone calnexin and ERp57 bind to the TAP-tapasin complex to generate an intermediate. These interactions are independent of the N-linked glycan of tapasin, but require its transmembrane and/or cytoplasmic domain. This intermediate complex binds MHC class I-β2m dimers, an event accompanied by the loss of calnexin and the acquisition of calreticulin, generating the MHC class I loading complex. Peptide binding then induces the dissociation of MHC class I-β2m dimers, which can be transported to the cell surface.

Ribosomal protein L2 is involved in the association of the ribosomal subunits, tRNA binding to A and P sites and peptidyl transfer

Ribosomal proteins L2, L3 and L4, together with the 23S RNA, are the main candidates for catalyzing peptide bond formation on the 50S subunit. That L2 is evolutionarily highly conserved led us to perform a thorough functional analysis with reconstituted 50S particles either lacking L2 or harboring a mutated L2. L2 does not play a dominant role in the assembly of the 50S subunit or in the fixation of the 3′‐ends of the tRNAs at the peptidyl‐transferase center. However, it is absolutely required for the association of 30S and 50S subunits and is strongly involved in tRNA binding to both A and P sites, possibly at the elbow region of the tRNAs. Furthermore, while the conserved histidyl residue 229 is extremely important for peptidyl‐transferase activity, it is apparently not involved in other measured functions. None of the other mutagenized amino acids (H14, D83, S177, D228, H231) showed this strong and exclusive participation in peptide bond formation. These results are used to examine critically the proposed direct involvement of His229 in catalysis of peptide synthesis.

Thiol oxidation and reduction in MHC-restricted antigen processing and presentation

Major histocompatibility complex (MHC) class I molecules are assembled in the endoplasmic reticulum (ER) as a trimer of the class I heavy chain, Β2 microglobulin (Β2m), and a short peptide. Assembly occurs in a complex with additional noncovalently associated proteins, which include the thiol oxidoreductase, ERp57. This molecule facilitates the formation of the correct disulfide bonds in glycoproteins as they fold in the ER and may play a key role in assembling a stable MHC class I-peptide complex. In the endocytic pathway, reduction of protein disulfide bonds is important for the generation of MHC class II-peptide complexes. This process is catalyzed by a γ-interferon-inducible thiol reductase (GILT). The possible requirement for catalysis of disulfide bond formation in MHC class I-restricted antigen processing and the known requirement for disulfide bond reduction in MHC class II-restricted antigen processing present interesting examples of the adaptation of cellular “housekeeping” functions to facilitate immune responses.

Mapping proteins of the 50S subunit from Escherichia coli ribosomes.

Mapping of protein positions in the ribosomal subunits was first achieved for the 30S subunit by means of neutron scattering about 15 years ago. Since the 50S subunit is almost twice as large as the 30S subunit and consists of more proteins, it was difficult to apply classical contrast variation techniques for the localisation of the proteins. Polarisation dependent neutron scattering (spin-contrast variation) helped to overcome this restriction. Here a map of 14 proteins within the 50S subunit from Escherichia coli ribosomes is presented including the proteins L17 and L20 that are not present in archeal ribosomes. The results are compared with the recent crystallographic map of the 50S subunit from the archea Haloarcula marismortui.

Architecture of the E.coli 70S ribosome

Abstract The 70S ribosome from E.coli was analysed by neutron scattering focusing on the shape and the internal protein-RNA-distribution of the complex. Measurements on selectively deuterated 70S particles and free 30S and 50S subunits applying conventional contrast variation and proton-spin contrast-variation resulted in a total of 42 scattering curves. Processing the data on the basis of the spherical harmonic technique, a four-phase model for the 70S ribosome could be generated, which describes the shape of the particle as well as the protein-and the RNA-moieties of each subunit at about 35 A resolution.

In situ structure determination of the ribosomal protein L14 in the large 50S subunit of Escherichia coli

Polarized neutron scattering from dynamically polarized nuclear spin targets has become a method of macromolecular structure research. The contrast created by substitution of the hydrogen isotope 1 H by 2 H is increased by almost a factor of three if polarized neutrons are scattered by polarized nuclear spins in the sample (spin contrast variation). Therefore, this method is also suitable to determine small or weakly contrasted labels. Proteins with a mass less than 1 wt.% of the background particle were localized. In this paper, an extension of our structural model for the 50S subunit by the in situ structure determination of the protein L14 is presented.