Significance: We analyzed 46 molecular structures of the yeast proteasome core particle (CP) by principal component analysis (PCA) and discovered two distinct configurations of the principal proteolytic subunit β5: the apo-cluster encompassing complexes with nonpeptidic ligands and the pep-cluster of complexes with peptidic ligands. Both configurations differ by a small domain motion and numerous slight global changes, thus enabling intersubunit communication. PCA was expanded to the mouse CP and revealed a striking difference between the constitutive CP and the immune CP. The former conforms to the yeast system and executes the structural change seen in yeast, although both immune apo and liganded CP classify into the pep configuration, a possible explanation for the generally higher activity of the immune proteasome.

Proprotein convertases (PCs) are highly specific proteases required for the proteolytic modification of many secreted proteins. An unbalanced activity of these enzymes is connected to pathologies like cancer, atherosclerosis, hypercholesterolaemia, and infectious diseases. Novel protein crystallographic structures of the prototypical PC family member furin in different functional states were determined to 1.8–2.0 Å. These, together with biochemical data and modeling by molecular dynamics calculations, suggest essential elements underlying its unusually high substrate specificity. Furin shows a complex activation mechanism and exists in at least four defined states: (i) the “off state,” incompatible with substrate binding as seen in the unliganded enzyme; (ii) the active “on state” seen in inhibitor-bound furin; and the respective (iii) calcium-free and (iv) calcium-bound forms. The transition from the off to the on state is triggered by ligand binding at subsites S1 to S4 and appears to underlie the preferential recognition of the four-residue sequence motif of furin. The molecular dynamics simulations of the four structural states reflect the experimental observations in general and provide approximations of the respective stabilities. Ligation by calcium at the PC-specific binding site II influences the active-site geometry and determines the rotamer state of the oxyanion hole-forming Asn295, and thus adds a second level of the activity modulation of furin. The described crystal forms and the observations of different defined functional states may foster the development of new tools and strategies for pharmacological intervention targeting furin. Significance The prototypical proprotein convertase furin proteolytically activates many precursor proteins and is essential for cellular homeostasis of the cell. Furin and other proprotein convertase are also associated with a number of diseases, including cancer, atherosclerosis, hypercholesterolaemia, and infectious diseases. A hallmark of furin and its physiologic function is its stringent specificity for polybasic substrates. Here we describe several structural states of the protein by X-ray crystallography and further characterize them by molecular dynamics simulations. The transition between these conformers is triggered by substrate binding and calcium ligation, respectively, and helps explain the stringent specificity of furin and its functional features. These studies may pave the way for novel strategies of structure-guided inhibitor development.

Dipeptidyl peptidases 8 and 9 are intracellular N-terminal dipeptidyl peptidases (preferentially postproline) associated with pathophysiological roles in immune response and cancer biology. While the DPP family member DPP4 is extensively characterized in molecular terms as a validated therapeutic target of type II diabetes, experimental 3D structures and ligand-/substrate-binding modes of DPP8 and DPP9 have not been reported. In this study we describe crystal and molecular structures of human DPP8 (2.5 Å) and DPP9 (3.0 Å) unliganded and complexed with a noncanonical substrate and a small molecule inhibitor, respectively. Similar to DPP4, DPP8 and DPP9 molecules consist of one β-propeller and α/β hydrolase domain, forming a functional homodimer. However, they differ extensively in the ligand binding site structure. In intriguing contrast to DPP4, where liganded and unliganded forms are closely similar, ligand binding to DPP8/9 induces an extensive rearrangement at the active site through a disorder-order transition of a 26-residue loop segment, which partially folds into an α-helix (R-helix), including R160/133, a key residue for substrate binding. As vestiges of this helix are also seen in one of the copies of the unliganded form, conformational selection may contributes to ligand binding. Molecular dynamics simulations support increased flexibility of the R-helix in the unliganded state. Consistently, enzyme kinetics assays reveal a cooperative allosteric mechanism. DPP8 and DPP9 are closely similar and display few opportunities for targeted ligand design. However, extensive differences from DPP4 provide multiple cues for specific inhibitor design and development of the DPP family members as therapeutic targets or antitargets.