Structure of Signal Peptide Peptidase A with C‑Termini Bound in the Active Sites: Insights into Specificity, Self-Processing, and Regulation

Bacterial signal peptide peptidase A (SppA) is a membrane-bound enzyme that utilizes a serine/lysine catalytic dyad mechanism to cleave remnant signal peptides within the cellular membrane. <i>Bacillus subtilis</i> SppA (SppA_BS) oligomerizes into a homo-octameric dome-shaped complex with eight active sites, located at the interface between each protomer. In this study, we show that SppA_BS self-processes its own C-termini. We have determined the crystal structure of a proteolytically stable fragment of SppA_BSK199A that has its C-terminal peptide bound in each of the eight active sites, creating a perfect circle of peptides. Substrate specificity pockets S1, S3, and S2′ are identified and accommodate C-terminal residues Tyr331, Met329, and Tyr333, respectively. Tyr331 at the P1 position is conserved among most <i>Bacillus</i> species. The structure reveals that the C-terminus binds within the substrate-binding grooves in an antiparallel β-sheet fashion. We show, by C-terminal truncations, that the C-terminus is not essential for oligomeric assembly. Kinetic analysis shows that a synthetic peptide corresponding to the C-terminus of SppA_BS competes with a fluorometric peptide substrate for the SppA_BS active site. A model is proposed for how the C-termini of SppA may function in the regulation of this membrane-bound self-compartmentalized protease

Structural Characterization of the C3 Domain of Cardiac Myosin Binding Protein C and Its Hypertrophic Cardiomyopathy-Related R502W Mutant

Human cardiac myosin binding protein C (cMyBP-C), a thick filament protein found within the sarcomere of cardiac muscle, regulates muscle contraction and is essential for proper muscle function. Hypertrophic cardiomyopathy (HCM), a genetic disease affecting 1 in 500 people, is the major cause of death in young athletes. It is caused by genetic mutations within sarcomeric proteins. Forty-two percent of the HCM-related mutations are found in cMyBP-C. Here we present the nuclear magnetic resonance-derived structural ensembles of the wild-type cMyBP-C C3 domain and its HCM-related R502W mutant. The C3 domain adopts an immunoglobulin-like fold, and mutation of the exposed Arg502 to a tryptophan does not perturb its structure, dynamics, or stability. However, the R502W mutation does alter the predicted electrostatic properties of the C3 domain. We hypothesize that this mutation, and other HCM-linked mutations found within the same domain, may directly disrupt the interaction of cMyBP-C with other sarcomeric proteins

Synthesis and Characterization of the Arylomycin Lipoglycopeptide Antibiotics and the Crystallographic Analysis of Their Complex with Signal Peptidase

Glycosylation of natural products, including antibiotics, often plays an important role in determining their physical properties and their biological activity, and thus their potential as drug candidates. The arylomycin class of antibiotics inhibits bacterial type I signal peptidase and is comprised of three related series of natural products with a lipopeptide tail attached to a core macrocycle. Previously, we reported the total synthesis of several A series derivatives, which have unmodified core macrocycles, as well as B series derivatives, which have a nitrated macrocycle. We now report the synthesis and biological evaluation of lipoglycopeptide arylomycin variants whose macrocycles are glycosylated with a deoxy-α-mannose substituent, and also in some cases hydroxylated. The synthesis of the derivatives bearing each possible deoxy-α-mannose enantiomer allowed us to assign the absolute stereochemistry of the sugar in the natural product and also to show that while glycosylation does not alter antibacterial activity, it does appear to improve solubility. Crystallographic structural studies of a lipoglycopeptide arylomycin bound to its signal peptidase target reveal the molecular interactions that underlie inhibition and also that the mannose is directed away from the binding site into solvent which suggests that other modifications may be made at the same position to further increase solubility and thus reduce protein binding and possibly optimize the pharmacokinetics of the scaffold