Stoichiometry of HLA Class II-Invariant Chain Oligomers Norbert Koch1*, Martin Zacharias2, Angelika Ko¨ nig1, Sebastian Temme1, Ju¨ rgen Neumann1, Sebastian Springer3 1 Division of Immunobiology, Institute of Genetics, University of Bonn, Bonn, Germany, 2 Physik Department, Technische Universita¨t Mu¨ nchen, Munich, Germany, 3 Molecular Life Science Center, Jacobs University Bremen, Bremen, Germany Abstract Background: The HLA gene complex encodes three class II isotypes, DR, DQ, and DP. HLA class II molecules are peptide receptors that present antigens for recognition by T lymphocytes. In antigen presenting cells, the assembly of matched a and b subunits to heterodimers is chaperoned by invariant chain (Ii). Ii forms a homotrimer with three binding sites for class II heterodimers. The current model of class II and Ii structure states that three ab heterodimers bind to an Ii trimer. Methology/Principal Findings: We have now analyzed the composition and size of the complexes of class II and Ii using epitope tagged class II subunits and density gradient experiments. We show here that class II-Ii oligomers consist of one class II heterodimer associated with one Ii trimer, such that the DR, DQ and DP isotypes are contained within separate complexes with Ii. Conclusion/Significance: We propose a structural model of the class II-Ii oligomer and speculate that the pentameric class II-Ii complex is bent towards the cell membrane, inhibiting the binding of additional class II heterodimers to Ii. Citation: Koch N, Zacharias M, Ko¨ nig A, Temme S, Neumann J, et al. (2011) Stoichiometry of HLA Class II-Invariant Chain Oligomers. PLoS ONE 6(2): e17257. doi:10.1371/journal.pone.0017257 Editor: Niels Caˆmara, Universidade de Sao Paulo, Brazil Received August 3, 2010; Accepted January 27, 2011; Published February 22, 2011 Copyright: 2011 Koch et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a grant from the Deutsche Forschungsgemeinschaft Ko 810/6. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: norbert.koch@uni-bonn.de Introduction Major histocompatibility complex (MHC) class II (MHCII) peptide receptors are sensors on antigen presenting cells (APC) that alert the adaptive immune system to the presence of foreign antigens by presenting them to CD4+ T cells. To achieve presentation of a large variety of antigenic peptides, the human MHC (human lymphocyte antigen, HLA) contains three isotypic loci, DP, DQ, and DR, each of which encode the a and b subunits that form heterodimeric MHCII receptors. An extremely high polymorphism of the MHCII genes creates a large diversity of the receptors. Following biosynthesis, the MHCII subunits associate with the chaperone invariant chain (Ii). Ii forms a homotrimer (Ii3) [1–3], and MHCII glycoproteins bind to a sequence of Ii that occupies the peptide binding groove of the ab heterodimer [4–6]. Additional interaction sites of MHCII and Ii have been demonstrated [7–11]. The homotrimeric structure of Ii would suggest that three MHCII heterodimers assemble with an Ii trimer to form a nonameric complex, Ii3(ab)3. Indeed, this assumption was supported by chemical crosslinking experiments with MHCII complexes that showed a high molecular weight band with an apparent size of 260 kDa [12–13]. The oligomeric assembly of MHCII and Ii is a prerequisite for intracellular transport to endosomes, followed by degradation of Ii and subsequent acquisition of exogenous peptides [14]. One possible consequence of the nonamer model is that one complex could contain different DP, DQ, or DR isotypes bound to an Ii homotrimer. Because of technical difficulties involved in distinguishing the individual isotype subunits, this obvious prediction has not been conclusively tested to date. Remarkably, initial experiments in mice have shown exclusive co-isolation of Ii with the murine MHCII isotypes H-2A or H-2E, but no co- isolation of these two isotypes with each other [15]. Based on this observation, we have now examined whether indeed DP, DQ, and DR are found in the same oligomer with Ii. In contrast to the MHCII heterodimers, Ii has a short cellular half-life [16–17]. Apparently as a compensation, Ii is synthesized at a significantly higher rate than MHCII, which leads to a large excess of the Ii polypeptide in the endoplasmic reticulum (ER) [18]. Since a stochastic assembly of MHCII subunits with Ii would therefore yield mostly pentameric complexes (with one MHCII heterodimer per Ii3), the formation of Ii3(ab)3 nonamers would require cooperative binding of MHCII ab heterodimers to Ii3. However, in vitro studies with soluble MHCII and Ii have suggested that complex formation is not sufficiently cooperative [19]. In recent years, novel methods have been employed to assess the stoichiometry of the murine cd T cell receptor and of the B cell antigen receptor [20–21]. These reports have yielded unpredicted results regarding the ratio of the antigen receptors to their associated signalling molecules and have thus led to the revision of previous models. In this work, we have employed epitope-tagged MHCII molecules to identify individual subunits of MHCII-Ii oligomers and to examine their stoichiometry. In transfected APCs, we have also studied the association of the tagged MHCII subunits with endogenous MHCII chains. We find that DP, DQ, and DR molecules are not co-isolated PLoS ONE | www.plosone.org 1 February 2011 | Volume 6 | Issue 2 | e17257