The major histocompatibility complex (MHC) class II–associated invariant chain (Ii) regulates intracellular trafficking and peptide loading of MHC class II molecules. Such loading occurs after endosomal degradation of the invariant chain to a ∼3-kD peptide termed CLIP (class II–associated invariant chain peptide). Cathepsins L and S have both been implicated in degradation of Ii to CLIP in thymus and peripheral lymphoid organs, respectively. However, macrophages from mice deficient in both cathepsins S and L can process Ii and load peptides onto MHC class II dimers normally. Both processes are blocked by a cysteine protease inhibitor, indicating the involvement of an additional Ii-processing enzyme(s). Comparison of cysteine proteases expressed by macrophages with those found in splenocytes and dendritic cells revealed two enzymes expressed exclusively in macrophages, cathepsins Z and F. Recombinant cathepsin Z did not generate CLIP from Ii–MHC class II complexes, whereas cathepsin F was as efficient as cathepsin S in CLIP generation. Inhibition of cathepsin F activity and MHC class II peptide loading by macrophages exhibited similar specificity and activity profiles. These experiments show that cathepsin F, in a subset of antigen presenting cells (APCs), can efficiently degrade Ii. Different APCs can thus use distinct proteases to mediate MHC class II maturation and peptide loading.

Rhomboids are intramembrane proteases that use a catalytic dyad of serine and histidine for proteolysis. They are conserved in both prokaryotes and eukaryotes and regulate cellular processes as diverse as intercellular signalling, parasitic invasion of host cells, and mitochondrial morphology. Their widespread biological significance and consequent medical potential provides a strong incentive to understand the mechanism of these unusual enzymes for identification of specific inhibitors. In this study, we describe the structure of Escherichia coli rhomboid GlpG covalently bound to a mechanism‐based isocoumarin inhibitor. We identify the position of the oxyanion hole, and the S1‐ and S2′‐binding subsites of GlpG, which are the key determinants of substrate specificity. The inhibitor‐bound structure suggests that subtle structural change is sufficient for catalysis, as opposed to large changes proposed from previous structures of unliganded GlpG. Using bound inhibitor as a template, we present a model for substrate binding at the active site and biochemically test its validity. This study provides a foundation for a structural explanation of rhomboid specificity and mechanism, and for inhibitor design.