Crystallographic studies of Rhizomucor miehei aspartic proteinase and its pepstatin A complex

The crystal structures of Rhizomucor miehei aspartic proteinase (RMP) and its pepstatin A complex have been determined at 2.15 A and 2.7 A, respectively. The structure of the native enzyme was refined to a crystallographic R-factor of 21.5% (R-free = 28.1%). RMP contains two domains that consist predominantly of β-sheets. A large substrate binding cleft is clearly visible between the two domains, and the two catalytic residues Asp38 and Asp237 are located in the middle of the cleft with a water molecule bridging the carboxyl groups of Asp38 and Asp237. It is proposed that the optimal pH of each aspartic proteinase is determined by the electrostatic potential at the active site, which, in turn, is determined by the positions and orientations of all the residues near the active site. RMP is the most glycosylated and most thermally stable among the aspartic proteinases. It is proposed that the highly flexible carbohydrates act as heat reservoirs to stabilize the conformation of RMP and thereby provide the enzyme with high thermal stability. Three-dimensional structural and sequence alignments of RMP with other aspartic proteinases suggest that RMP and Mucor pusillus aspartic proteinase (MPP) diverged from the main stream of aspartic proteinases at an early stage of evolution. The structure of the RMP-pepstatin A complex was refined to a crystallographic R-value of 19.3% (R-free = 28.0%). In the final model, a pepstatin A molecule fits into the large substrate-binding cleft between the two domains of RMP in an extended conformation up to the alanine residue at the P2\sp\prime position. The statine residue at the "P3\sp\prime-P4\sp\prime" position forms an inverse γ-turn (P3\sp\prime-P1\sp\prime), with its leucyl side chain binding into the S1\sp\prime subsite. The inhibitor interacts with the residues of the substrate binding pocket by either hydrogen bonds or hydrophobic interactions, or both

Architecturally diverse proteins converge on an analogous mechanism to inactivate Uracil-DNA glycosylase

Uracil-DNA glycosylase (UDG) compromises the replication strategies of diverse viruses from unrelated lineages. Virally encoded proteins therefore exist to limit, inhibit or target UDG activity for proteolysis. Viral proteins targeting UDG, such as the bacteriophage proteins ugi, and p56, and the HIV-1 protein Vpr, share no sequence similarity, and are not structurally homologous. Such diversity has hindered identification of known or expected UDG-inhibitory activities in other genomes. The structural basis for UDG inhibition by ugi is well characterized; yet, paradoxically, the structure of the unbound p56 protein is enigmatically unrevealing of its mechanism. To resolve this conundrum, we determined the structure of a p56 dimer bound to UDG. A helix from one of the subunits of p56 occupies the UDG DNA-binding cleft, whereas the dimer interface forms a hydrophobic box to trap a mechanistically important UDG residue. Surprisingly, these p56 inhibitory elements are unexpectedly analogous to features used by ugi despite profound architectural disparity. Contacts from B-DNA to UDG are mimicked by residues of the p56 helix, echoing the role of ugi’s inhibitory beta strand. Using mutagenesis, we propose that DNA mimicry by p56 is a targeting and specificity mechanism supporting tight inhibition via hydrophobic sequestration

Kinetic and Crystallographic Studies of Drug-Resistant Mutants of HIV-1 Protease: Insights into the Drug Resistance Mechanisms

HIV-1 protease (PR) inhibitors (PIs) are important anti-HIV drugs for the treatment of AIDS and have shown great success in reducing mortality and prolonging the life of HIV-infected individuals. However, the rapid development of drug resistance is one of the major factors causing the reduced effectiveness of PIs. Consequently, various drug resistant mutants of HIV-1 PR have been extensively studied to gain insight into the mechanisms of drug resistance. In this study, the crystal structures, dimer stabilities, and kinetics data have been analyzed for wild type PR and over 10 resistant mutants including PRL24I, PRI32V, PRM46L, PRG48V, PRI50V, PRF53L, PRI54V, PRI54M, PRG73S and PRL90M. These mutations lie in varied structural regions of PR: adjacent to the active site, in the inhibitor binding site, the flap or at protein surface. The enzymatic activity and inhibition were altered in mutant PR to various degrees. Crystal structures of the mutants complexed with a substrate analog inhibitor or drugs indinavir, saquinavir and darunavir were determined at resolutions of 0.84 – 1.50 Å. Each mutant revealed distinct structural changes, which are usually located at the mutated residue, the flap and inhibitor binding sites. Moreover, darunavir was shown to bind to PR at a new site on the flap surface in PRI32V and PRM46L. The existence of this additional inhibitor binding site may explain the high effectiveness of darunavir on drug resistant mutants. Moreover, the unliganded structure PRF53L had a wider separation at the tips of the flaps than in unliganded wild type PR. The absence of flap interactions in PRF53L suggests a novel mechanism for drug resistance. Therefore, this study enhanced our understanding of the role of individual residues in the development of drug resistance and the structural basis of drug resistance mechanisms. Atomic resolution crystal structures are valuable for the design of more potent protease inhibitors to overcome the drug resistance problem

Interdependence of Inhibitor Recognition in HIV-1 Protease

Molecular recognition is a highly interdependent process. Subsite couplings within the active site of proteases are most often revealed through conditional amino acid preferences in substrate recognition. However, the potential effect of these couplings on inhibition and thus inhibitor design is largely unexplored. The present study examines the interdependency of subsites in HIV-1 protease using a focused library of protease inhibitors, to aid in future inhibitor design. Previously a series of darunavir (DRV) analogs was designed to systematically probe the S1\u27 and S2\u27 subsites. Co-crystal structures of these analogs with HIV-1 protease provide the ideal opportunity to probe subsite interdependency. All-atom molecular dynamics simulations starting from these structures were performed and systematically analyzed in terms of atomic fluctuations, intermolecular interactions, and water structure. These analyses reveal that the S1\u27 subsite highly influences other subsites: the extension of the hydrophobic P1\u27 moiety results in 1) reduced van der Waals contacts in the P2\u27 subsite, 2) more variability in the hydrogen bond frequencies with catalytic residues and the flap water, and 3) changes in the occupancy of conserved water sites both proximal and distal to the active site. In addition, one of the monomers in this homodimeric enzyme has atomic fluctuations more highly correlated with DRV than the other monomer. These relationships intricately link the HIV-1 protease subsites and are critical to understanding molecular recognition and inhibitor binding. More broadly, the interdependency of subsite recognition within an active site requires consideration in the selection of chemical moieties in drug design; this strategy is in contrast to what is traditionally done with independent optimization of chemical moieties of an inhibitor

The prosequence of procaricain forms an α-helical domain that prevents access to the substrate-binding cleft

AbstractBackground Cysteine proteases are involved in a variety of cellular processes including cartilage degradation in arthritis, the progression of Alzheimer's disease and cancer invasion: these enzymes are therefore of immense biological importance. Caricain is the most basic of the cysteine proteases found in the latex of Carica papaya. It is a member of the papain superfamily and is homologous to other plant and animal cysteine proteases. Caricain is naturally expressed as an inactive zymogen called procaricain. The inactive form of the protease contains an inhibitory proregion which consists of an additional 106 N-terminal amino acids; the proregion is removed upon activation.Results The crystal structure of procaricain has been refined to 3.2 å resolution; the final model consists of three non-crystallographically related molecules. The proregion of caricain forms a separate globular domain which binds to the C-terminal domain of mature caricain. The proregion also contains an extended polypeptide chain which runs through the substrate-binding cleft, in the opposite direction to that of the substrate, and connects to the N terminus of the mature region. The mature region does not undergo any conformational change on activation.Conclusions We conclude that the rate-limiting step in the in vitro activation of procaricain is the dissociation of the prodomain, which is then followed by proteolytic cleavage of the extended polypeptide chain of the proregion. The prodomain provides a stable scaffold which may facilitate the folding of the C-terminal lobe of procaricain

Structural and Functional Characterization of the Endosome-associated Deubiquitinating Enzyme AMSH

The endosomal sorting complexes required for transport (ESCRT) machinery is a ubiquitin-dependent molecular mechanism made of up of four individual complexes: ESCRT-0, -I, -II, III, that is necessary for regulating the degradation of cell surface receptors directed towards the lysosome. Not only are the ESCRTs implicated in endosomal sorting and trafficking of proteins, its members also have roles in other important biological processes such as: cytokinesis, HIV budding, transcriptional regulation, and autophagy. As a function of its involvement in several processes throughout the cell, the ESCRT machinery is implicated in a wide variety of diseases including cancer, neurological disease, bacterial infections, cardiovascular disease, and retroviral infection. Proteins marked for lysosomal degradation (cargo) are first ubiquitinated, and then, shuttled in a sequential mechanism through the complexes. In the last step, ubiquitin is removed from the cargo, which is subsequently encapsulated into intralumenal vesicles (ILV) that will ultimately be transported to and fuse with lysosome, degrading and recycling its contents. Deubiquitination is the removal of ubiquitin, catalyzed by deubiquitinating enzymes (DUBs). The human ESCRT machinery recruits two DUBs: AMSH (associated molecule with a Src homology 3 (SH3) domain of signal transducing adaptor molecule (STAM) or simply, STAM-binding protein (STAMBP)), and UBPY/USP8 (ubiquitin specific protease 8). Both AMSH and USP8 have the same ESCRT-recognition domains facilitating recruitment to ESCRT-0 and ESCRT-III. The Saccharmyces cerevisiae (S. cerevisiae) version of the ESCRT complex employs only one DUB, Doa4 (degradation of alpha 4) that serves to recycle ubiquitin at ESCRT-III, just prior to ILV formation. Therefore, it is not fully understood why the human ESCRT system requires the function of both AMSH and USP8. The focus of this thesis is to understand the role of AMSH recruitment at ESCRT-0 with hopes of providing further insight into its role within the ESCRT complex. In doing so, I crystallized and determined the structure of catalytic domain of AMSH. Using this structure, I structurally and thermodynamically compared AMSH to the homologous protein, AMSH-LP. Secondly, I characterized AMSH kinetically by introducing individual point mutations within the catalytic domain and carried out a detailed kinetic analysis to understand the catalytic mechanism of AMSH. Finally, using a combination of biophysical and biochemical experiments, I investigated how AMSH is recruited and recognized at ESCRT-0. My studies show that AMSH is structurally identical to AMSH-LP, however, thermodynamically less stable. Also, AMSH has exquisite specificity for Lys63-linked ubiquitin chains because it recognizes a three-residue sequence within its proximal ubiquitin-binding site. Furthermore, two residues within the distal ubiquitin-binding site (Thr313 and Glu316) play significant roles within AMSH\u27s catalytic mechanism, one of which, Thr313, is mutated to Ile in children with microcephaly capillary malformation (MIC-CAP) syndrome. Finally, I proposed a mechanism for how the activity of AMSH is stimulated at ESCRT-0 in which the proximal ubiquitin is held by the ubiquitin-interacting motif (UIM) from STAM (ESCRT-0), while the enzyme holds the distal ubiquitin, thus stabilizing the chain, enhancing the enzyme\u27s activity. From this mechanism, I assigned a role for AMSH at ESCRT-0 in which the enzyme facilitates the transfer of cargo from ESCRT-0 to the subsequent complexes. These data taken together further supports that AMSH has an important, specific, and non-redundant function within the ESCRT machinery

The dimer-monomer equilibrium of SARS-CoV-2 main protease is affected by small molecule inhibitors

none14noThe maturation of coronavirus SARS-CoV-2, which is the etiological agent at the origin of the COVID-19 pandemic, requires a main protease Mpro to cleave the virus-encoded polyproteins. Despite a wealth of experimental information already available, there is wide disagreement about the Mpro monomer-dimer equilibrium dissociation constant. Since the functional unit of Mpro is a homodimer, the detailed knowledge of the thermodynamics of this equilibrium is a key piece of information for possible therapeutic intervention, with small molecules interfering with dimerization being potential broad-spectrum antiviral drug leads. In the present study, we exploit Small Angle X-ray Scattering (SAXS) to investigate the structural features of SARS-CoV-2 Mpro in solution as a function of protein concentration and temperature. A detailed thermodynamic picture of the monomer-dimer equilibrium is derived, together with the temperature-dependent value of the dissociation constant. SAXS is also used to study how the Mpro dissociation process is affected by small inhibitors selected by virtual screening. We find that these inhibitors affect dimerization and enzymatic activity to a different extent and sometimes in an opposite way, likely due to the different molecular mechanisms underlying the two processes. The Mpro residues that emerge as key to optimize both dissociation and enzymatic activity inhibition are discussed.openSilvestrini L.; Belhaj N.; Comez L.; Gerelli Y.; Lauria A.; Libera V.; Mariani P.; Marzullo P.; Ortore M.G.; Palumbo Piccionello A.; Petrillo C.; Savini L.; Paciaroni A.; Spinozzi F.Silvestrini, L.; Belhaj, N.; Comez, L.; Gerelli, Y.; Lauria, A.; Libera, V.; Mariani, P.; Marzullo, P.; Ortore, M. G.; Palumbo Piccionello, A.; Petrillo, C.; Savini, L.; Paciaroni, A.; Spinozzi, F