A matrix-focused structure-activity and binding site flexibility study of quinolinol inhibitors of botulinum neurotoxin serotype A

Our initial discovery of 8-hydroxyquinoline inhibitors of BoNT/A and separation/testing of enantiomers of one of the more active leads indicated considerable flexibility in the binding site. We designed a limited study to investigate this flexibility and probe structure-activity relationships; utilizing the Betti reaction, a 36 compound matrix of quinolinol BoNT/A LC inhibitors was developed using three 8-hydroxyquinolines, three heteroaromatic amines, and four substituted benzaldehydes. This study has revealed some of the most effective quinolinol-based BoNT/A inhibitors to date, with 7 compounds displaying IC₅₀ values ?1 μM and 11 effective at ?2 μM in an ex vivo assay.     

Association of botulinum neurotoxin serotype A light chain with plasma membrane-bound SNAP-25

The Clostridium botulinum neurotoxins (BoNTs) cleave SNARE proteins, which inhibit binding and thus fusion of neurotransmitter vesicles to the plasma membrane of peripheral neurons. BoNTs comprise an N-terminal light chain (LC) and C-terminal heavy chain, which are linked by a disulfide bond. There are seven serotypes (A-G) of BoNTs based upon immunological neutralization. Although the binding and entry of BoNT/A into neurons has been subjected to considerable investigation, the intracellular events that allow BoNT/A to efficiently cleave SNAP-25 within neurons is less well understood. Earlier studies showed that intracellular LC/A bound to the plasma membrane of neurons. In this study, intracellular LC/A is shown to directly bind SNAP-25 on the plasma membrane. Solid phase binding showed that the N-terminal residues of LC/A bound residues 80-110 of SNAP-25, which was also observed in cultured neurons. Association of the N-terminal 8 amino acids of LC/A and residues 80-110 of SNAP-25 also enhanced substrate cleavage. These findings explain how LC/A associates with SNAP-25 on the plasma membrane and provide a basis for LC/A cleavage of SNAP-25 within the SNARE complex.     

Molecular characterization of the protease from Clostridium botulinum serotype C responsible for nicking in botulinum neurotoxin complex

A protease was purified from the culture medium of Clostridium botulinum serotype C strain Stockholm (C-St). The purified protease belonged to the cysteine protease family based on assays for enzyme inhibitors, activators and kinetic parameters. The protease formed a binary complex consisting of 41- and 17-kDa proteins held together non-covalently. The DNA sequence encoding the protease gene was shown to be a single open reading frame of 1593 nucleotides, predicting 530 amino acid residues including a signal peptide. The N-terminal region of the native enzyme underwent further proteolytic modification after processing by a signal peptidase. The protease introduced intermolecular cleavage into an intact single chain botulinum neurotoxin (BoNT) at a specific site. Homology modeling and docking simulation of C-St BoNT and C-St protease demonstrated that the specific nicking-site of the BoNT appears to fit into the deep pocket in the active site of the protease.     

Identification of the synaptic vesicle glycoprotein 2 receptor binding site in botulinum neurotoxin A

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release by hydrolysing SNARE proteins. The most important serotype BoNT/A employs the synaptic vesicle glycoprotein 2 (SV2) isoforms A-C as neuronal receptors. Here, we identified their binding site by blocking SV2 interaction using monoclonal antibodies with characterised epitopes within the cell binding domain (H_C). The site is located on the backside of the conserved ganglioside binding pocket at the interface of the H_CC and H_CN subdomains. The dimension of the binding pocket was characterised in detail by site directed mutagenesis allowing the development of potent inhibitors as well as modifying receptor binding properties.     

Linkers for improved cleavage of fusion proteins with an engineered alpha-lytic protease.

Addition of an N-terminal fusion partner can greatly aid the expression and purification of a recombinant protein in Escherichia coli. We investigated two genetically engineered proteases designed to remove the fusion partner after the protein of interest has been expressed. Recombinant human insulin-like growth factor-II (hIGF-II) has been produced from E. coli-derived fusion proteins using a novel enzymatic cleavage system that uses a mutant of alpha-lytic protease. Initially, two potential fusion protein linkers were designed, Pro-Ala-Pro-His (PAPH) and Pro-Ala-Pro-Met (PAPM), and were tested as substrates in the form of synthetic dodecapeptides. Using mass spectrometry and reverse-phase HPLC, the position of cleavage was confirmed and the kinetics of synthetic peptide cleavage were examined. Use of the linkers in hIGF-II fusion proteins produced in E. coli was then evaluated. The fusion proteins constructed consist of the first 11 amino acids of porcine growth hormone linked N-terminally to hIGF-II by six amino acids that include the dipeptide Val-Asn followed by a variable tetrapeptide protease cleavage motif. Mass spectrometry and N-terminal sequencing confirmed that proteolytic cleavage of the fusion proteins had occurred at the predicted sites. Using the fusion proteins as substrates, the cleavage of the rationally designed motifs by the alpha-lytic protease mutant was compared. The fusion protein containing the motif PAPM had a k(cat)/K(M) ratio indicating a 1.6-fold preference over the PAPH fusion protein for cleavage by this enzyme. Furthermore, when hIGF-II fusion proteins containing the designed cleavable linkers were processed with the engineered alpha-lytic protease, they gave greatly improved yields of native hIGF-II compared to an analogous fusion protein cleaved by H64A subtilisin. Comparison of the peptide and protein cleavage studies shows that the efficient proteolysis of the cleavage motifs is an inherent property of the designed sequences and is not determined by secondary or tertiary structure in the fusion proteins.     

In silico analysis of ORF1ab in coronavirus HKU1 genome reveals a unique putative cleavage site of coronavirus HKU1 3C-like protease

Recently we have described the discovery and complete genome sequence of a novel coronavirus associated with pneumonia, coronavirus HKU1 (CoV-HKU1). In this study, a detailed in silico analysis of the ORF1ab, encoding the 7,182-amino acid replicase polyprotein in the CoV-HKU1 genome showed that the replicase polyprotein of CoV-HKU1 is cleaved by its papain-like proteases and 3C-like protease (3CL(pro)) into 16 polypeptides homologous to the corresponding polypeptides in other coronaviruses. Surprisingly, analysis of the putative cleavage sites of the 3CL(pro) revealed a unique putative cleavage site. In all known coronaviruses, the P1 positions at the cleavage sites of the 3CL(pro) are occupied by glutamine. This is also observed in CoV-HKU1, except for one site at the junction between nsp10 (helicase) and nsp11 (member of exonuclease family), where the P1 position is occupied by histidine. This amino acid substitution is due to a single nucleotide mutation in the CoV-HKU1 genome, CAG/A to CAT. This probably represents a novel cleavage site because the same mutation was consistently observed in CoV-HKU1 sequences from multiple specimens of different patients; the P2 and P1'-P12' positions of this cleavage site are consistent between CoV-HKU1 and other coronaviruses; and as the helicase is one of the most conserved proteins in coronaviruses, cleavage between nsp10 and nsp11 should be an essential step for the generation of the mature functional helicase. Experiments, including purification and C-terminal amino acid sequencing of the CoV-HKU1 helicase and trans-cleavage assays of the CoV-HKU1 3CL(pro) will confirm the presence of this novel cleavage site.     

Investigation of the induced-fit mechanism and catalytic activity of the human cytomegalovirus protease homodimer via molecular dynamics simulations

Human cytomegalovirus (HCMV) is a highly species-specific DNA virus infecting up to 80% of the general population. The viral genome contains the open reading frame UL80, which encodes the full-length 80 kDa HCMV serine protease and its substrate. Full-length HCMV protease is composed of an N-terminal 256-amino-acid proteolytic domain, called assemblin, a linker region, and a C-terminal structural domain, the assembly protein precursor. Biochemical studies have shown that dimerization activates assemblin because of an induced stabilization of the oxyanion hole (Arg166). Thus, we performed here molecular dynamics (MD) simulations on HCMV protease models to study the induced-fit mechanism of the enzyme upon the binding of substrates and peptidyl inhibitors, and structural and energetic factors that are responsible for the catalytic activity of the enzyme dimer. Long and stable trajectories were obtained for the models of the monomeric and dimeric states, free in solution and bound to a peptidyl-activated carbonyl inhibitor, with very good agreement between theoretical and experimental results. Our results suggest that HCMV protease is indeed a novel example of serine protease that operates by an induced-fit mechanism. Also, in agreement with mutagenesis studies, our MD simulations suggest that the dimeric form is necessary to activate the enzyme because of an induced stabilization of the oxyanion hole.     

Important functional role of residue x of the presenilin GxGD protease active site motif for APP substrate cleavage specificity and substrate selectivity of γ‐secretase

γ‐Secretase plays a central role in the generation of the Alzheimer disease‐causing amyloid β‐peptide (Aβ) from the β‐amyloid precursor protein (APP) and is thus a major Alzheimer′s disease drug target. As several other γ‐secretase substrates including Notch1 and CD44 have crucial signaling functions, an understanding of the mechanism of substrate recognition and cleavage is key for the development of APP selective γ‐secretase‐targeting drugs. The γ‐secretase active site domain in its catalytic subunit presenilin (PS) 1 has been implicated in substrate recognition/docking and cleavage. Highly critical in this process is its GxGD active site motif, whose invariant glycine residues cannot be replaced without causing severe functional losses in substrate selection and/or cleavage efficiency. Here, we have investigated the contribution of the less well characterized residue x of the motif (L383 in PS1) to this function. Extensive mutational analysis showed that processing of APP was overall well‐tolerated over a wide range of hydrophobic and hydrophilic mutations. Interestingly, however, most L383 mutants gave rise to reduced levels of Aβ_37–39 species, and several increased the pathogenic Aβ_42/43 species. Several of the Aβ_42/43‐increasing mutants severely impaired the cleavages of Notch1 and CD44 substrates, which were not affected by any other L383 mutation. Our data thus establish an important, but compared with the glycine residues of the motif, overall less critical functional role for L383. We suggest that L383 and the flanking glycine residues form a spatial arrangement in PS1 that is critical for docking and/or cleavage of different γ‐secretase substrates.     

A novel auto-cleavage assay for studying mutational effects on the active site of severe acute respiratory syndrome coronavirus 3C-like protease

The 3C-like protease (3CLpro) of severe acute respiratory syndrome (SARS) has been proposed as an attractive target for drug design. His41 and Cys145 were essential for the active site as the principal catalytic residues. In this study, we mutated the two sites, expressed four resulting mutants in Escherichia coli and characterized. All mutants showed undetectable activity in trans-cleavage assay. In addition, we introduced a 31-mer peptide containing an auto-cleavage site to the N-terminal of the proteases and found the peptide could be cleaved efficiently by 3CLsc itself, but, among the four mutants, only the mutant Cys145-->Ser showed residual activity as detected by the auto-cleavage assay. The data supported the proposition unequivocally that SARS-CoV 3CLpro was a member of serine proteases involving His41 and Cys145 residues at the active site. The auto-cleavage assay also provided a sensitive and reliable compensation to the traditional trans-cleavage assay.     

A novel approach for enhancing the catalytic efficiency of a protease at low temperature: Reduction in substrate inhibition by chemical modification

The alkaline protease, savinase was chemically modified to enhance the productivity of the enzyme at low temperatures on a complex polymeric protein (azocasein) substrate. At 5 and 15°C, savinase modified with ficol or dextran hydrolyzed fivefold more azocasein than the unmodified savinase. Kinetic studies showed that the catalytic improvements are associated with changes in uncompetitive substrate inhibition with K_i values of modified savinases sixfold higher than the unmodified savinase. Modeling of small‐angle scattering data indicates that two substrate molecules bind on opposing sides of the enzyme. The combined kinetic and structural data indicate that the polysaccharide modifier sterically blocks the allosteric site and reduces substrate inhibition. In contrast to the properties of cold‐active enzymes that generally manifest as low activation enthalpy and high flexibility, this study shows that increased activity and productivity at low temperature can be achieved by reducing uncompetitive substrate inhibition, and that this can be achieved using chemical modification with an enzyme in a commercial enzyme‐formulation. Biotechnol. Bioeng. 2009;103: 676–686. © 2009 Wiley Periodicals, Inc.