Project description:The anticoagulant serpin, protein Z-dependent protease inhibitor (ZPI), is catalytically activated by its cofactor, protein Z (PZ), to regulate the function of blood coagulation factor Xa on membrane surfaces. The X-ray structure of the ZPI-PZ complex has shown that PZ binds to a unique site on ZPI centered on helix G. In the present study, we show by Ala-scanning mutagenesis of the ZPI-binding interface, together with native PAGE and kinetic analyses of PZ binding to ZPI, that Tyr240 and Asp293 of ZPI are crucial hot spots for PZ binding. Complementary studies with protein Z-protein C chimeras show the importance of both pseudocatalytic and EGF2 domains of PZ for the critical ZPI interactions. To understand how PZ acts catalytically, we analyzed the interaction of reactive loop-cleaved ZPI (cZPI) with PZ and determined the cZPI X-ray structure. The cZPI structure revealed changes in helices A and G of the PZ-binding site relative to native ZPI that rationalized an observed 6-fold loss in PZ affinity and PZ catalytic action. These findings identify the key determinants of catalytic activation of ZPI by PZ and suggest novel strategies for ameliorating hemophilic states through drugs that disrupt the ZPI-PZ interaction.
Project description:DegP is a heat shock protein from high temperature requirement protease A family, which reacts to the environmental stress conditions in an ATP independent way. The objective of the present analysis emerged from the temperature dependent functional diversity of DegP between chaperonic and protease activities at temperatures below and above 28 °C, respectively. DegP is a multimeric protein and the minimal functional unit, DegP-trimer, is of great importance in understanding the DegP pathway. The structural aspects of DegP-trimer with respect to temperature variation have been studied using molecular dynamics simulations (for 100 ns) and principal component analysis to highlight the temperature dependent dynamics facilitating its functional diversity. The DegP-trimer revealed a pronounced dynamics at both 280 and 320 K, when compared to the dynamics observed at 300 K. The LA loop is identified as the highly flexible region during dynamics and at extreme temperatures, the residues 46-80 of LA loop express a flip towards right (at 280) and left ( at 320 K) with respect to the fixed ?-sheet connecting the LA loop of protease for which Phe46 acts as one of the key residues. Such dynamics of LA loop facilitates inter-monomeric interaction with the PDZ1 domain of the neighbouring monomer and explains its active participation when DegP exists as trimer. Hence, the LA loop mediated dynamics of DegP-trimer is expected to provide further insight into the temperature dependent dynamics of DegP towards the understanding of its assembly and functional diversity in the presence of substrate.
Project description:Substrate-bound structures of AAA+ protein translocases reveal a conserved asymmetric spiral staircase architecture wherein a sequential ATP hydrolysis cycle drives hand-over-hand substrate translocation. However, this configuration is unlikely to represent the full conformational landscape of these enzymes, as biochemical studies suggest distinct conformational states depending on the presence or absence of substrate. Here, we used cryo-electron microscopy to determine structures of the Yersinia pestis Lon AAA+ protease in the absence and presence of substrate, uncovering the mechanistic basis for two distinct operational modes. In the absence of substrate, Lon adopts a left-handed, "open" spiral organization with autoinhibited proteolytic active sites. Upon the addition of substrate, Lon undergoes a reorganization to assemble an enzymatically active, right-handed "closed" conformer with active protease sites. These findings define the mechanistic principles underlying the operational plasticity required for processing diverse protein substrates.
Project description:DegP, a member of the highly conserved HtrA family, performs quality-control degradation of misfolded proteins in the periplasm of gram-negative bacteria and is required for high-temperature survival of Escherichia coli. Substrate binding transforms DegP from an inactive oligomer containing two trimers into active polyhedral cages, typically containing four or eight trimers. Although these observations suggest a causal connection, we show that cage assembly and proteolytic activation can be uncoupled. Indeed, DegP variants that remain trimeric, hexameric, or dodecameric in the presence or absence of substrate still display robust and positively cooperative substrate degradation in vitro and, most importantly, sustain high-temperature bacterial growth as well as the wild-type enzyme. Our results support a model in which substrate binding converts inactive trimers into proteolytically active trimers, and simultaneously leads to cage assembly by enhancing binding of PDZ1 domains in one trimer to PDZ2' domains in neighboring trimers. Thus, both processes depend on substrate binding, but they can be uncoupled without loss of biological function. We discuss potential coupling mechanisms and why cage formation may have evolved if it is not required for DegP proteolysis.
Project description:Rising antibiotic resistance urgently begs for novel targets and strategies for antibiotic discovery. Here, we report that over-activation of the periplasmic DegP protease, a member of the highly conserved HtrA family, can be a viable strategy for antibiotic development. We demonstrate that tripodal peptidyl compounds that mimic DegP-activating lipoprotein variants allosterically activate DegP and inhibit the growth of an Escherichia coli strain with a permeable outer membrane in a DegP-dependent fashion. Interestingly, these compounds inhibit bacterial growth at a temperature at which DegP is not essential for cell viability, mainly by over-proteolysis of newly synthesized proteins. Co-crystal structures show that the peptidyl arms of the compounds bind to the substrate-binding sites of DegP. Overall, our results represent an intriguing example of killing bacteria by activating a non-essential enzyme, and thus expand the scope of antibiotic targets beyond the traditional essential proteins or pathways.
Project description:Omptins constitute a unique family of outer membrane proteases that are widespread in Enterobacteriaceae. The plasminogen activator (Pla) of Yersinia pestis is an omptin family member that is very important for development of both bubonic and pneumonic plague. The physiological function of Pla is to cleave (activate) human plasminogen to form the plasma protease plasmin. Uniquely, lipopolysaccharide (LPS) is essential for the catalytic activity of all omptins, including Pla. Why omptins require LPS for enzymatic activity is unknown. Here, we report the co-crystal structure of LPS-free Pla in complex with the activation loop peptide of human plasminogen, its natural substrate. The structure shows that in the absence of LPS, the peptide substrate binds deep within the active site groove and displaces the nucleophilic water molecule, providing an explanation for the dependence of omptins on LPS for enzymatic activity.
Project description:Proper activation of protein phosphatase 2A (PP2A) catalytic subunit is central for the complex PP2A regulation and is crucial for broad aspects of cellular function. The crystal structure of PP2A bound to PP2A phosphatase activator (PTPA) and ATP?S reveals that PTPA makes broad contacts with the structural elements surrounding the PP2A active site and the adenine moiety of ATP. PTPA-binding stabilizes the protein fold of apo-PP2A required for activation, and orients ATP phosphoryl groups to bind directly to the PP2A active site. This allows ATP to modulate the metal-binding preferences of the PP2A active site and utilize the PP2A active site for ATP hydrolysis. In vitro, ATP selectively and drastically enhances binding of endogenous catalytic metal ions, which requires ATP hydrolysis and is crucial for acquisition of pSer/Thr-specific phosphatase activity. Furthermore, both PP2A- and ATP-binding are required for PTPA function in cell proliferation and survival. Our results suggest novel mechanisms of PTPA in PP2A activation with structural economy and a unique ATP-binding pocket that could potentially serve as a specific therapeutic target.
Project description:Plant metacaspases mediate programmed cell death in development, biotic and abiotic stresses, damage-induced immune response, and resistance to pathogen attack. Most metacaspases require Ca2+ for their activation and substrate processing. However, the Ca2+-dependent activation mechanism remains elusive. Here we report the crystal structures of Metacaspase 4 from Arabidopsis thaliana (AtMC4) that modulates Ca2+-dependent, damage-induced plant immune defense. The AtMC4 structure exhibits an inhibitory conformation in which a large linker domain blocks activation and substrate access. In addition, the side chain of Lys225 in the linker domain blocks the active site by sitting directly between two catalytic residues. We show that the activation of AtMC4 and cleavage of its physiological substrate involve multiple cleavages in the linker domain upon activation by Ca2+. Our analysis provides insight into the Ca2+-dependent activation of AtMC4 and lays the basis for tuning its activity in response to stresses for engineering of more sustainable crops for food and biofuels.
Project description:The combination of thiol protease activity and calmodulin-like EF-hands is a feature unique to the calpains. The regulatory mechanisms governing calpain activity are complex, and the nature of the Ca(2+)-induced switch between inactive and active forms has remained elusive in the absence of structural information. We describe here the 2.6 A crystal structure of m-calpain in the Ca(2+)-free form, which illustrates the structural basis for the inactivity of calpain in the absence of Ca(2+). It also reveals an unusual thiol protease fold, which is associated with Ca(2+)-binding domains through heterodimerization and a C(2)-like beta-sandwich domain. Strikingly, the structure shows that the catalytic triad is not assembled, indicating that Ca(2+)-binding must induce conformational changes that re-orient the protease domains to form a functional active site. The alpha-helical N-terminal anchor of the catalytic subunit does not occupy the active site but inhibits its assembly and regulates Ca(2+)-sensitivity through association with the regulatory subunit. This Ca(2+)-dependent activation mechanism is clearly distinct from those of classical proteases.
Project description:The active RNA-dependent RNA polymerase of poliovirus, 3Dpol, is generated by cleavage of the 3CDpro precursor protein, a protease that has no polymerase activity despite containing the entire polymerase domain. By intentionally disrupting a known and persistent crystal packing interaction, we have crystallized the poliovirus polymerase in a new space group and solved the complete structure of the protein at 2.0 A resolution. It shows that the N-terminus of fully processed 3Dpol is buried in a surface pocket where it makes hydrogen bonds that act to position Asp238 in the active site. Asp238 is an essential residue that selects for the 2' OH group of substrate rNTPs, as shown by a 2.35 A structure of a 3Dpol-GTP complex. Mutational, biochemical, and structural data further demonstrate that 3Dpol activity is exquisitely sensitive to mutations at the N-terminus. This sensitivity is the result of allosteric effects where the structure around the buried N-terminus directly affects the positioning of Asp238 in the active site.