Publications by authors named "Lakshmi C Wijeyewickrema"

38 Publications

Mapping the binding site of C1-inhibitor for polyanion cofactors.

Mol Immunol 2020 10 24;126:8-13. Epub 2020 Jul 24.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia; ARC Centre of Excellence in Advanced Molecular Imaging, La Trobe University, Melbourne, Victoria 3086, Australia. Electronic address:

The serpin, C1-inhibitor (also known as SERPING1), plays a vital anti-inflammatory role in the body by controlling pro-inflammatory pathways such as complement and coagulation. The inhibitor's action is enhanced in the presence of polyanionic cofactors, such as heparin and polyphosphate, by increasing the rate of association with key enzymes such as C1s of the classical pathway of complement. The cofactor binding site of the serpin has never been mapped. Here we show that residues Lys284, Lys285 and Arg287 of C1-inhibitor play key roles in binding heparin and delivering the rate enhancement seen in the presence of polyanions and thus most likely represent the key cofactor binding residues for the serpin. We also show that simultaneous binding of the anion binding site of C1s by the polyanion is required to deliver the rate enhancement. Finally, we have shown that it is unlikely that the two positively charged zones of C1-inhibitor and C1s interact in the encounter complex between molecules as ablation of the charged zones did not in itself deliver a rate enhancement as might have been expected if the zones interacted. These insights provide crucial information as to the mechanism of action of this key serpin in the presence and absence of cofactor molecules.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molimm.2020.06.018DOI Listing
October 2020

Protease-associated import systems are widespread in Gram-negative bacteria.

PLoS Genet 2019 10 15;15(10):e1008435. Epub 2019 Oct 15.

Infection and Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, Australia.

Bacteria have evolved sophisticated uptake machineries in order to obtain the nutrients required for growth. Gram-negative plant pathogens of the genus Pectobacterium obtain iron from the protein ferredoxin, which is produced by their plant hosts. This iron-piracy is mediated by the ferredoxin uptake system (Fus), a gene cluster encoding proteins that transport ferredoxin into the bacterial cell and process it proteolytically. In this work we show that gene clusters related to the Fus are widespread in bacterial species. Through structural and biochemical characterisation of the distantly related Fus homologues YddB and PqqL from Escherichia coli, we show that these proteins are analogous to components of the Fus from Pectobacterium. The membrane protein YddB shares common structural features with the outer membrane ferredoxin transporter FusA, including a large extracellular substrate binding site. PqqL is an active protease with an analogous periplasmic localisation and iron-dependent expression to the ferredoxin processing protease FusC. Structural analysis demonstrates that PqqL and FusC share specific features that distinguish them from other members of the M16 protease family. Taken together, these data provide evidence that protease associated import systems analogous to the Fus are widespread in Gram-negative bacteria.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1008435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6793856PMC
October 2019

Determination of the crystal structure and substrate specificity of ananain.

Biochimie 2019 Nov 12;166:194-202. Epub 2019 Jul 12.

Department of Biochemistry & Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Australia; ARC Centre of Excellence for Advanced Molecular Imaging. Melbourne, Australia. Electronic address:

Ananain (EC 3.4.22.31) accounts for less than 10% of the total enzyme in the crude pineapple stem extract known as bromelain, yet yields the majority of the proteolytic activity of bromelain. Despite a high degree of sequence identity between ananain and stem bromelain, the most abundant bromelain cysteine protease, ananain displays distinct chemical properties, substrate preference and inhibitory profile compared to stem bromelain. A tripeptidyl substrate library (REPLi) was used to further characterize the substrate specificity of ananain and identified an optimal substrate for cleavage by ananain. The optimal tripeptide, PLQ, yielded a high k/K value of 1.7 x 106 Ms, with cleavage confirmed to occur after the Gln residue. Crystal structures of unbound ananain and an inhibitory complex of ananain and E-64, solved at 1.73 and 1.98 Å, respectively, revealed a geometrically flat and open S1 subsite for ananain. This subsite accommodates diverse P1 substrate residues, while a narrow and deep hydrophobic pocket-like S2 subsite would accommodate a non-polar P2 residue, such as the preferred Leu residue observed in the specificity studies. A further illustration of the atomic interactions between E-64 and ananain explains the high inhibitory efficiency of E-64 toward ananain. These data reveal the first in depth structural and functional data for ananain and provide a basis for further study of the natural properties of the enzyme.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biochi.2019.07.011DOI Listing
November 2019

Molecular basis for the folding of β-helical autotransporter passenger domains.

Nat Commun 2018 04 11;9(1):1395. Epub 2018 Apr 11.

Research School of Biology, Australian National University, Canberra, ACT, 0200, Australia.

Bacterial autotransporters comprise a C-terminal β-barrel domain, which must be correctly folded and inserted into the outer membrane to facilitate translocation of the N-terminal passenger domain to the cell exterior. Once at the surface, the passenger domains of most autotransporters are folded into an elongated β-helix. In a cellular context, key molecules catalyze the assembly of the autotransporter β-barrel domain. However, how the passenger domain folds into its functional form is poorly understood. Here we use mutational analysis on the autotransporter Pet to show that the β-hairpin structure of the fifth extracellular loop of the β-barrel domain has a crucial role for passenger domain folding into a β-helix. Bioinformatics and structural analyses, and mutagenesis of a homologous autotransporter, suggest that this function is conserved among autotransporter proteins with β-helical passenger domains. We propose that the autotransporter β-barrel domain is a folding vector that nucleates folding of the passenger domain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-03593-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5895577PMC
April 2018

The Structural Basis for Complement Inhibition by Gigastasin, a Protease Inhibitor from the Giant Amazon Leech.

J Immunol 2017 12 23;199(11):3883-3891. Epub 2017 Oct 23.

Department of Biochemistry and Genetics and Australian Research Council Centre of Excellence in Advanced Molecular Imaging, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia;

Complement is crucial to the immune response, but dysregulation of the system causes inflammatory disease. Complement is activated by three pathways: classical, lectin, and alternative. The classical and lectin pathways are initiated by the C1r/C1s (classical) and MASP-1/MASP-2 (lectin) proteases. Given the role of complement in disease, there is a requirement for inhibitors to control the initiating proteases. In this article, we show that a novel inhibitor, gigastasin, from the giant Amazon leech, potently inhibits C1s and MASP-2, whereas it is also a good inhibitor of MASP-1. Gigastasin is a poor inhibitor of C1r. The inhibitor blocks the active sites of C1s and MASP-2, as well as the anion-binding exosites of the enzymes via sulfotyrosine residues. Complement deposition assays revealed that gigastasin is an effective inhibitor of complement activation in vivo, especially for activation via the lectin pathway. These data suggest that the cumulative effects of inhibiting both MASP-2 and MASP-1 have a greater effect on the lectin pathway than the more potent inhibition of only C1s of the classical pathway.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1700158DOI Listing
December 2017

Recruitment of Human C1 Esterase Inhibitor Controls Complement Activation on Blood Stage Merozoites.

J Immunol 2017 06 8;198(12):4728-4737. Epub 2017 May 8.

Infection and Immunity Division, The Walter and Eliza Hall Institute, Parkville, Victoria 3052, Australia;

The complement system is a front-line defense system that opsonizes and lyses invading pathogens. To survive, microbes exposed to serum must evade the complement response. To achieve this, many pathogens recruit soluble human complement regulators to their surfaces and hijack their regulatory function for protection from complement activation. C1 esterase inhibitor (C1-INH) is a soluble regulator of complement activation that negatively regulates the classical and lectin pathways of complement to protect human tissue from aberrant activation. In this article, we show that merozoites, the invasive form of blood stage malaria parasites, actively recruit C1-INH to their surfaces when exposed to human serum. We identified MSP3.1, a member of the merozoite surface protein 3 family of merozoite surface proteins, as the direct interaction partner. When bound to the merozoite surface, C1-INH retains its ability to complex with and inhibit C1s, MASP1, and MASP2, the activating proteases of the complement cascade. merozoites that lack MSP3.1 showed a marked reduction in C1-INH recruitment and increased C3b deposition on their surfaces. However, these ΔMSP3.1 merozoites exhibit enhanced invasion of RBCs in the presence of active complement. This study characterizes an immune-evasion strategy used by malaria parasites and highlights the complex relationship between merozoites and the complement system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1700067DOI Listing
June 2017

Polyphosphate is a novel cofactor for regulation of complement by a serpin, C1 inhibitor.

Blood 2016 09 23;128(13):1766-76. Epub 2016 Jun 23.

Centre for Blood Research, Department of Medicine, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada;

The complement system plays a key role in innate immunity, inflammation, and coagulation. The system is delicately balanced by negative regulatory mechanisms that modulate the host response to pathogen invasion and injury. The serpin, C1-esterase inhibitor (C1-INH), is the only known plasma inhibitor of C1s, the initiating serine protease of the classical pathway of complement. Like other serpin-protease partners, C1-INH interaction with C1s is accelerated by polyanions such as heparin. Polyphosphate (polyP) is a naturally occurring polyanion with effects on coagulation and complement. We recently found that polyP binds to C1-INH, prompting us to consider whether polyP acts as a cofactor for C1-INH interactions with its target proteases. We show that polyP dampens C1s-mediated activation of the classical pathway in a polymer length- and concentration-dependent manner by accelerating C1-INH neutralization of C1s cleavage of C4 and C2. PolyP significantly increases the rate of interaction between C1s and C1-INH, to an extent comparable to heparin, with an exosite on the serine protease domain of the enzyme playing a major role in this interaction. In a serum-based cell culture system, polyP significantly suppressed C4d deposition on endothelial cells, generated via the classical and lectin pathways. Moreover, polyP and C1-INH colocalize in activated platelets, suggesting that their interactions are physiologically relevant. In summary, like heparin, polyP is a naturally occurring cofactor for the C1s:C1-INH interaction and thus an important regulator of complement activation. The findings may provide novel insights into mechanisms underlying inflammatory diseases and the development of new therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2016-02-699561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5043130PMC
September 2016

Structural basis for substrate specificity of Helicobacter pylori M17 aminopeptidase.

Biochimie 2016 Feb 1;121:60-71. Epub 2015 Dec 1.

Infection and Immunity Program, Monash Biomedical Discovery Institute and Department of Microbiology, Monash University, Clayton, Victoria, Australia; Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia. Electronic address:

The M17 aminopeptidase from the carcinogenic gastric bacterium Helicobacter pylori (HpM17AP) is an important housekeeping enzyme involved in catabolism of endogenous and exogenous peptides. It is implicated in H. pylori defence against the human innate immune response and in the mechanism of metronidazole resistance. Bestatin inhibits HpM17AP and suppresses H. pylori growth. To address the structural basis of catalysis and inhibition of this enzyme, we have established its specificity towards the N-terminal amino acid of peptide substrates and determined the crystal structures of HpM17AP and its complex with bestatin. The position of the D-phenylalanine moiety of the inhibitor with respect to the active-site metal ions, bicarbonate ion and with respect to other M17 aminopeptidases suggested that this residue binds to the S1 subsite of HpM17AP. In contrast to most characterized M17 aminopeptidases, HpM17AP displays preference for L-Arg over L-Leu residues in peptide substrates. Compared to very similar homologues from other bacteria, a distinguishing feature of HpM17AP is a hydrophilic pocket at the end of the S1 subsite that is likely to accommodate the charged head group of the L-Arg residue of the substrate. The pocket is flanked by a sodium ion (not present in M17 aminopeptidases that show preference for L-Leu) and its coordinating water molecules. In addition, the structure suggests that variable loops at the entrance to, and in the middle of, the substrate-binding channel are important determinants of substrate specificity of M17 aminopeptidases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biochi.2015.11.021DOI Listing
February 2016

Protein unfolding is essential for cleavage within the α-helix of a model protein substrate by the serine protease, thrombin.

Biochimie 2016 Mar 25;122:227-34. Epub 2015 Sep 25.

Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, 3800, Australia.

Proteolysis has a critical role in transmitting information within a biological system and therefore an important element of biology is to determine the subset of proteins amenable to proteolysis. Until recently, it has been thought that proteases cleave native protein substrates only within solvent exposed loops, but recent evidence indicates that cleavage sites located within α-helices can also be cleaved by proteases, despite the conformation of this secondary structure being generally incompatible with binding into an active site of a protease. In this study, we address the mechanism by which a serine endopeptidase, thrombin, recognizes and cleaves a target sequence located within an α-helix. Thrombin was able to cleave a model substrate, protein G, within its α-helix when a suitable cleavage sequence for the enzyme was introduced into this region. However, structural data for the complex revealed that thrombin was not perturbing the structure of the α-helix, thus it was not destabilizing the helix in order to allow it to fit within its active site. This indicated that thrombin was only cleaving within the α-helix when it was in an unfolded state. In support of this, the introduction of destabilizing mutations within the protein increased the efficiency of cleavage by the enzyme. Our data suggest that a folded α-helix cannot be proteolytically cleaved by thrombin, but the species targeted are the unfolded conformations of the native state ensemble.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biochi.2015.09.021DOI Listing
March 2016

Investigation of the mechanism of interaction between Mannose-binding lectin-associated serine protease-2 and complement C4.

Mol Immunol 2015 Oct 27;67(2 Pt B):287-93. Epub 2015 Jun 27.

Department of Biochemistry & Genetics, La Trobe Institute of Molecular Science, La Trobe University, Melbourne, Victoria, 3086, Australia.

The interaction between mannose-binding lectin [MBL]-associated serine protease-2 (MASP-2) and its first substrate, C4 is crucial to the lectin pathway of complement, which is vital for innate host immunity, but also involved in a number of inflammatory diseases. Recent data suggests that two areas outside of the active site of MASP-2 (so-called exosites) are crucial for efficient cleavage of C4: one at the junction of the two complement control protein (CCP) domains of the enzyme and the second on the serine protease (SP) domain. Here, we have further investigated the roles of each of these exosites in the binding and cleavage of C4. We have found that both exosites are required for high affinity binding and efficient cleavage of the substrate protein. Within the SP domain exosite, we have shown here that two arginine residues are most important for high affinity binding and efficient cleavage of C4. Finally, we show that the CCP domain exosite appears to play the major role in the initial interaction with C4, whilst the SP domain exosite plays the major role in a secondary conformational change between the two proteins required to form a high affinity complex. This data has provided new insights into the binding and cleavage of C4 by MASP-2, which may be useful in the design of molecules that modulate this important interaction required to activate the lectin pathway of complement.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molimm.2015.06.011DOI Listing
October 2015

Scabies mite inactive serine proteases are potent inhibitors of the human complement lectin pathway.

PLoS Negl Trop Dis 2014 May 22;8(5):e2872. Epub 2014 May 22.

Infectious Diseases Division, QIMR Berghofer Medical Research Institute, Brisbane, Australia.

Scabies is an infectious skin disease caused by the mite Sarcoptes scabiei and has been classified as one of the six most prevalent epidermal parasitic skin diseases infecting populations living in poverty by the World Health Organisation. The role of the complement system, a pivotal component of human innate immunity, as an important defence against invading pathogens has been well documented and many parasites have an arsenal of anti-complement defences. We previously reported on a family of scabies mite proteolytically inactive serine protease paralogues (SMIPP-Ss) thought to be implicated in host defence evasion. We have since shown that two family members, SMIPP-S D1 and I1 have the ability to bind the human complement components C1q, mannose binding lectin (MBL) and properdin and are capable of inhibiting all three human complement pathways. This investigation focused on inhibition of the lectin pathway of complement activation as it is likely to be the primary pathway affecting scabies mites. Activation of the lectin pathway relies on the activation of MBL, and as SMIPP-S D1 and I1 have previously been shown to bind MBL, the nature of this interaction was examined using binding and mutagenesis studies. SMIPP-S D1 bound MBL in complex with MBL-associated serine proteases (MASPs) and released the MASP-2 enzyme from the complex. SMIPP-S I1 was also able to bind MBL in complex with MASPs, but MASP-1 and MASP-2 remained in the complex. Despite these differences in mechanism, both molecules inhibited activation of complement components downstream of MBL. Mutagenesis studies revealed that both SMIPP-Ss used an alternative site of the molecule from the residual active site region to inhibit the lectin pathway. We propose that SMIPP-Ss are potent lectin pathway inhibitors and that this mechanism represents an important tool in the immune evasion repertoire of the parasitic mite and a potential target for therapeutics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pntd.0002872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4031079PMC
May 2014

Total synthesis of homogeneous variants of hirudin P6: a post-translationally modified anti-thrombotic leech-derived protein.

Angew Chem Int Ed Engl 2014 Apr 11;53(15):3947-51. Epub 2014 Mar 11.

School of Chemistry, The University of Sydney, Sydney, NSW 2006 (Australia) http://sydney.edu.au/science/chemistry/∼payne/index.

Hirudin P6 is a leech-derived anti-thrombotic protein which possesses two post-translational modifications, O-glycosylation and tyrosine sulfation. In this study we report the ligation-based synthesis of a library of hirudin P6 proteins possessing homogeneous glycosylation and sulfation modifications. The nature of the modifications incorporated was shown to have a drastic effect on inhibition against both the fibrinogenolytic and amidolytic activities of thrombin and thus highlights a potential means for attenuating the biological activity of the protein.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/anie.201310777DOI Listing
April 2014

A molecular basis for the association of the HLA-DRB1 locus, citrullination, and rheumatoid arthritis.

J Exp Med 2013 Nov 4;210(12):2569-82. Epub 2013 Nov 4.

Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia.

Rheumatoid arthritis (RA) is strongly associated with the human leukocyte antigen (HLA)-DRB1 locus that possesses the shared susceptibility epitope (SE) and the citrullination of self-antigens. We show how citrullinated aggrecan and vimentin epitopes bind to HLA-DRB1*04:01/04. Citrulline was accommodated within the electropositive P4 pocket of HLA-DRB1*04:01/04, whereas the electronegative P4 pocket of the RA-resistant HLA-DRB1*04:02 allomorph interacted with arginine or citrulline-containing epitopes. Peptide elution studies revealed P4 arginine-containing peptides from HLA-DRB1*04:02, but not from HLA-DRB1*04:01/04. Citrullination altered protease susceptibility of vimentin, thereby generating self-epitopes that are presented to T cells in HLA-DRB1*04:01(+) individuals. Using HLA-II tetramers, we observed citrullinated vimentin- and aggrecan-specific CD4(+) T cells in the peripheral blood of HLA-DRB1*04:01(+) RA-affected and healthy individuals. In RA patients, autoreactive T cell numbers correlated with disease activity and were deficient in regulatory T cells relative to healthy individuals. These findings reshape our understanding of the association between citrullination, the HLA-DRB1 locus, and T cell autoreactivity in RA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1084/jem.20131241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3832918PMC
November 2013

The molecular switches controlling the interaction between complement proteases of the classical and lectin pathways and their substrates.

Curr Opin Struct Biol 2013 Dec 7;23(6):820-7. Epub 2013 Aug 7.

Department of Biochemistry & Molecular Biology, Monash University, Clayton, Victoria 3800, Australia. Electronic address:

Complement represents a major bridge between the innate and adaptive immune systems of the body. It plays a vital role in host defences against pathogens, but has also been implicated in numerous inflammatory diseases. The system has been the subject of intensive research in recent times with a number of key structural insights into the functioning of the system. Here, we will give an overview of the activation of each pathway, following which recent developments in our understanding of the mechanisms governing the interaction between enzymes and substrates in the classical and lectin pathways in particular will be discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.sbi.2013.07.016DOI Listing
December 2013

The x-ray crystal structure of mannose-binding lectin-associated serine proteinase-3 reveals the structural basis for enzyme inactivity associated with the Carnevale, Mingarelli, Malpuech, and Michels (3MC) syndrome.

J Biol Chem 2013 Aug 21;288(31):22399-407. Epub 2013 Jun 21.

Department of Biochemistry & Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.

The mannose-binding lectin associated-protease-3 (MASP-3) is a member of the lectin pathway of the complement system, a key component of human innate and active immunity. Mutations in MASP-3 have recently been found to be associated with Carnevale, Mingarelli, Malpuech, and Michels (3MC) syndrome, a severe developmental disorder manifested by cleft palate, intellectual disability, and skeletal abnormalities. However, the molecular basis for MASP-3 function remains to be understood. Here we characterize the substrate specificity of MASP-3 by screening against a combinatorial peptide substrate library. Through this approach, we successfully identified a peptide substrate that was 20-fold more efficiently cleaved than any other identified to date. Furthermore, we demonstrated that mutant forms of the enzyme associated with 3MC syndrome were completely inactive against this substrate. To address the structural basis for this defect, we determined the 2.6-Å structure of the zymogen form of the G666E mutant of MASP-3. These data reveal that the mutation disrupts the active site and perturbs the position of the catalytic serine residue. Together, these insights into the function of MASP-3 reveal how a mutation in this enzyme causes it to be inactive and thus contribute to the 3MC syndrome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M113.483875DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3829330PMC
August 2013

A molecular switch governs the interaction between the human complement protease C1s and its substrate, complement C4.

J Biol Chem 2013 May 16;288(22):15821-9. Epub 2013 Apr 16.

Department of Biochemistry and Molecular Biology, Monash University, Clayton, Melbourne, Victoria 3800, Australia.

The complement system is an ancient innate immune defense pathway that plays a front line role in eliminating microbial pathogens. Recognition of foreign targets by antibodies drives sequential activation of two serine proteases, C1r and C1s, which reside within the complement Component 1 (C1) complex. Active C1s propagates the immune response through its ability to bind and cleave the effector molecule complement Component 4 (C4). Currently, the precise structural and biochemical basis for the control of the interaction between C1s and C4 is unclear. Here, using surface plasmon resonance, we show that the transition of the C1s zymogen to the active form is essential for C1s binding to C4. To understand this, we determined the crystal structure of a zymogen C1s construct (comprising two complement control protein (CCP) domains and the serine protease (SP) domain). These data reveal that two loops (492-499 and 573-580) in the zymogen serine protease domain adopt a conformation that would be predicted to sterically abrogate C4 binding. The transition from zymogen to active C1s repositions both loops such that they would be able to interact with sulfotyrosine residues on C4. The structure also shows the junction of the CCP1 and CCP2 domains of C1s for the first time, yielding valuable information about the exosite for C4 binding located at this position. Together, these data provide a structural explanation for the control of the interaction with C1s and C4 and, furthermore, point to alternative strategies for developing therapeutic approaches for controlling activation of the complement cascade.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M113.464545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3668739PMC
May 2013

Molecular determinants of the substrate specificity of the complement-initiating protease, C1r.

J Biol Chem 2013 May 15;288(22):15571-80. Epub 2013 Apr 15.

Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.

The serine protease, C1r, initiates activation of the classical pathway of complement, which is a crucial innate defense mechanism against pathogens and altered-self cells. C1r both autoactivates and subsequently cleaves and activates C1s. Because complement is implicated in many inflammatory diseases, an understanding of the interaction between C1r and its target substrates is required for the design of effective inhibitors of complement activation. Examination of the active site specificity of C1r using phage library technology revealed clear specificity for Gln at P2 and Ile at P1', which are found in these positions in physiological substrates of C1r. Removal of one or both of the Gln at P2 and Ile at P1' in the C1s substrate reduced the rate of C1r activation. Substituting a Gln residue into the P2 of the activation site of MASP-3, a protein with similar domain structure to C1s that is not normally cleaved by C1r, enabled efficient activation of this enzyme. Molecular dynamics simulations and structural modeling of the interaction of the C1s activation peptide with the active site of C1r revealed the molecular mechanisms that particularly underpin the specificity of the enzyme for the P2 Gln residue. The complement control protein domains of C1r also made important contributions to efficient activation of C1s by this enzyme, indicating that exosite interactions were also important. These data show that C1r specificity is well suited to its cleavage targets and that efficient cleavage of C1s is achieved through both active site and exosite contributions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M113.451757DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3668718PMC
May 2013

Assembly of the type II secretion system such as found in Vibrio cholerae depends on the novel Pilotin AspS.

PLoS Pathog 2013 Jan 10;9(1):e1003117. Epub 2013 Jan 10.

Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia.

The Type II Secretion System (T2SS) is a molecular machine that drives the secretion of fully-folded protein substrates across the bacterial outer membrane. A key element in the machinery is the secretin: an integral, multimeric outer membrane protein that forms the secretion pore. We show that three distinct forms of T2SSs can be distinguished based on the sequence characteristics of their secretin pores. Detailed comparative analysis of two of these, the Klebsiella-type and Vibrio-type, showed them to be further distinguished by the pilotin that mediates their transport and assembly into the outer membrane. We have determined the crystal structure of the novel pilotin AspS from Vibrio cholerae, demonstrating convergent evolution wherein AspS is functionally equivalent and yet structurally unrelated to the pilotins found in Klebsiella and other bacteria. AspS binds to a specific targeting sequence in the Vibrio-type secretins, enhances the kinetics of secretin assembly, and homologs of AspS are found in all species of Vibrio as well those few strains of Escherichia and Shigella that have acquired a Vibrio-type T2SS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1003117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542185PMC
January 2013

Identification of a catalytic exosite for complement component C4 on the serine protease domain of C1s.

J Immunol 2012 Sep 1;189(5):2365-73. Epub 2012 Aug 1.

Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.

The classical pathway of complement is crucial to the immune system, but it also contributes to inflammatory diseases when dysregulated. Binding of the C1 complex to ligands activates the pathway by inducing autoactivation of associated C1r, after which C1r activates C1s. C1s cleaves complement component C4 and then C2 to cause full activation of the system. The interaction between C1s and C4 involves active site and exosite-mediated events, but the molecular details are unknown. In this study, we identified four positively charged amino acids on the serine protease domain that appear to form a catalytic exosite that is required for efficient cleavage of C4. These residues are coincidentally involved in coordinating a sulfate ion in the crystal structure of the protease. Together with other evidence, this pointed to the involvement of sulfate ions in the interaction with the C4 substrate, and we showed that the protease interacts with a peptide from C4 containing three sulfotyrosine residues. We present a molecular model for the interaction between C1s and C4 that provides support for the above data and poses questions for future research into this aspect of complement activation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1201085DOI Listing
September 2012

Novel scabies mite serpins inhibit the three pathways of the human complement system.

PLoS One 2012 11;7(7):e40489. Epub 2012 Jul 11.

Infectious Diseases Program, Biology Department, Queensland Institute of Medical Research, Brisbane, Queensland, Australia.

Scabies is a parasitic infestation of the skin by the mite Sarcoptes scabiei that causes significant morbidity worldwide, in particular within socially disadvantaged populations. In order to identify mechanisms that enable the scabies mite to evade human immune defenses, we have studied molecules associated with proteolytic systems in the mite, including two novel scabies mite serine protease inhibitors (SMSs) of the serpin superfamily. Immunohistochemical studies revealed that within mite-infected human skin SMSB4 (54 kDa) and SMSB3 (47 kDa) were both localized in the mite gut and feces. Recombinant purified SMSB3 and SMSB4 did not inhibit mite serine and cysteine proteases, but did inhibit mammalian serine proteases, such as chymotrypsin, albeit inefficiently. Detailed functional analysis revealed that both serpins interfered with all three pathways of the human complement system at different stages of their activation. SMSB4 inhibited mostly the initial and progressing steps of the cascades, while SMSB3 showed the strongest effects at the C9 level in the terminal pathway. Additive effects of both serpins were shown at the C9 level in the lectin pathway. Both SMSs were able to interfere with complement factors without protease function. A range of binding assays showed direct binding between SMSB4 and seven complement proteins (C1, properdin, MBL, C4, C3, C6 and C8), while significant binding of SMSB3 occurred exclusively to complement factors without protease function (C4, C3, C8). Direct binding was observed between SMSB4 and the complement proteases C1s and C1r. However no complex formation was observed between either mite serpin and the complement serine proteases C1r, C1s, MASP-1, MASP-2 and MASP-3. No catalytic inhibition by either serpin was observed for any of these enzymes. In summary, the SMSs were acting at several levels mediating overall inhibition of the complement system and thus we propose that they may protect scabies mites from complement-mediated gut damage.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0040489PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3394726PMC
April 2013

Effect of O-glycosylation and tyrosine sulfation of leech-derived peptides on binding and inhibitory activity against thrombin.

Chem Commun (Camb) 2012 Feb 23;48(10):1547-9. Epub 2011 Nov 23.

School of Chemistry, The University of Sydney, NSW 2006, Australia.

Synthesis of sulfated and unsulfated (glyco)peptide fragments of Hirudin P6 (a potent anticoagulant from the leech Hirudinaria manillensis) is described. The effect of O-glycosylation and tyrosine sulfation on thrombin binding and peptidolytic activity was investigated, together with the inhibition of fibrinogen cleavage.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c1cc14773kDOI Listing
February 2012

Multiple domains of MASP-2, an initiating complement protease, are required for interaction with its substrate C4.

Mol Immunol 2012 Jan 8;49(4):593-600. Epub 2011 Nov 8.

Department of Biochemistry & Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.

The complement system is fundamental to both innate and adaptive immunity and can be initiated via the classical, lectin or alternative pathways. Cleavage of C4 by MASP-2, the initiating protease of the lectin pathway, is a crucial event in the activation of this pathway, preceding the eventual formation of the C3 convertase (C4bC2a) complex on the pathogen surface. Interactions required for the cleavage of C4 by MASP-2 are likely to be facilitated by the initial binding of C4 to an exosite on the protease. We have shown that both proteolytically active and catalytically inactive CCP1-CCP2-serine protease (CCP1-CCP2-SP) forms bind C4 with similar affinity. Interestingly, proteins containing the CCP1-CCP2 domains or the SP domain alone bound C4 with much lower affinity than the CCP1-CCP2-SP protein, suggesting that the CCP domains cooperate positively with the active site to mediate efficient binding and cleavage of C4. In addition, mutation of residue K342 to alanine in the CCP1 domain abolished binding to both C4 and C4b in its CCP1-CCP2 form, suggesting a key electrostatic role for this amino acid. The presented data indicates that all of the domains are required in order to mediate high affinity interaction with C4.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molimm.2011.10.006DOI Listing
January 2012

Discovery of amino acid motifs for thrombin cleavage and validation using a model substrate.

Biochemistry 2011 Dec 11;50(48):10499-507. Epub 2011 Nov 11.

Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.

Understanding the active site preferences of an enzyme is critical to the design of effective inhibitors and to gaining insights into its mechanisms of action on substrates. While the subsite specificity of thrombin is understood, it is not clear whether the enzyme prefers individual amino acids at each subsite in isolation or prefers to cleave combinations of amino acids as a motif. To investigate whether preferred peptide motifs for cleavage could be identified for thrombin, we exposed a phage-displayed peptide library to thrombin. The resulting preferentially cleaved substrates were analyzed using the technique of association rule discovery. The results revealed that thrombin selected for amino acid motifs in cleavage sites. The contribution of these hypothetical motifs to substrate cleavage efficiency was further investigated using the B1 IgG-binding domain of streptococcal protein G as a model substrate. Introduction of a P(2)-P(1)' LRS thrombin cleavage sequence within a major loop of the protein led to cleavage of the protein by thrombin, with the cleavage efficiency increasing with the length of the loop. Introduction of further P(3)-P(1) and P(1)-P(1)'-P(3)' amino acid motifs into the loop region yielded greater cleavage efficiencies, suggesting that the susceptibility of a protein substrate to cleavage by thrombin is influenced by these motifs, perhaps because of cooperative effects between subsites closest to the scissile peptide bond.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi201333gDOI Listing
December 2011

S1 pocket of a bacterially derived subtilisin-like protease underpins effective tissue destruction.

J Biol Chem 2011 Dec 11;286(49):42180-42187. Epub 2011 Oct 11.

Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800, Australia; Departments of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.

The ovine footrot pathogen, Dichelobacter nodosus, secretes three subtilisin-like proteases that play an important role in the pathogenesis of footrot through their ability to mediate tissue destruction. Virulent and benign strains of D. nodosus secrete the basic proteases BprV and BprB, respectively, with the catalytic domain of these enzymes having 96% sequence identity. At present, it is not known how sequence variation between these two putative virulence factors influences their respective biological activity. We have determined the high resolution crystal structures of BprV and BprB. These data reveal that that the S1 pocket of BprV is more hydrophobic but smaller than that of BprB. We show that BprV is more effective than BprB in degrading extracellular matrix components of the host tissue. Mutation of two residues around the S1 pocket of BprB to the equivalent residues in BprV dramatically enhanced its proteolytic activity against elastin substrates. Application of a novel approach for profiling substrate specificity, the Rapid Endopeptidase Profiling Library (REPLi) method, revealed that both enzymes prefer cleaving after hydrophobic residues (and in particular P1 leucine) but that BprV has more restricted primary substrate specificity than BprB. Furthermore, for P1 Leu-containing substrates we found that BprV is a significantly more efficient enzyme than BprB. Collectively, these data illuminate how subtle changes in D. nodosus proteases may significantly influence tissue destruction as part of the ovine footrot pathogenesis process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M111.298711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3234984PMC
December 2011

Serpins and the complement system.

Methods Enzymol 2011 ;499:55-75

Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Hungary.

C1-inhibitor (serpin G1) is a 105 kDa inhibitor which functions as a major antiinflammatory protein in the body. It has its effects via inhibition of the proteases of the complement system and contact system of coagulation, as well as several direct effects mediated by its unique highly glycosylated N-terminal domain. The serpin controls a number of different proteases very efficiently and for some of these the function is augmented by the cofactor, heparin. Here, we describe the preparation of human plasma and recombinant C1-inhibitor and the basic methods required for their characterization, using the complement enzyme C1s as an example of a target enzyme.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/B978-0-12-386471-0.00004-3DOI Listing
October 2011

Mannose-binding lectin serine proteases and associated proteins of the lectin pathway of complement: two genes, five proteins and many functions?

Biochim Biophys Acta 2012 Jan 6;1824(1):253-62. Epub 2011 Jun 6.

Department of Biochemistry & Molecular Biology, Monash University, Clayton, Victoria, Australia.

The lectin pathway of the complement system is activated following the binding of carbohydrate-based ligands by recognition molecules such as mannose-binding lectin (MBL) or ficolins. Engagement of the recognition molecules causes activation of associated MBL-associated serine proteases or MASPs, which in turn activate downstream complement molecules to activate the system. Two MASP genes are alternatively spliced during expression to yield 5 proteins, including three proteases (MASP-1, -2 and -3) and two truncated proteins, MAp19 and MAp44. Here we discuss what is currently known about these proteins in terms of their structure and function. MASP-2 is autoactivated following the initial binding events of the pathway and is able to subsequently activate the C4 and C2 substrates required to activate the rest of the pathway. MASP-1 is able to augment MASP-2 activation, but also appears to play other roles, although the physiological significance of these is not yet clear. The roles of the truncated Map19 and Map44 proteins and the MASP-3 protease are currently unknown. The proteases form an interesting sub-family of proteins that clearly should be the focus of future research in order to establish their biological roles. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbapap.2011.05.021DOI Listing
January 2012

The lysine-specific gingipain of Porphyromonas gingivalis : importance to pathogenicity and potential strategies for inhibition.

Adv Exp Med Biol 2011 ;712:15-29

Department of Biochemistry and Molecular Biology and CRC for Oral Health Sciences, Monash University, Clayton, Victoria, Australia.

Periodontitis is a disease affecting the supporting structures of the teeth. The most severe forms of the disease result in tooth loss and have recently been strongly associated with systemic diseases, including cardiovascular and lung diseases and cancer. The disease is caused by biofilms of predominantly anaerobic bacteria. A major pathogen associated with severe, adult forms of the disease is Porphyromonas gingivalis. This organism produces potent cysteine proteases known as gingipains, which have specificity for cleavage after arginine or lysine residues. The lysine-specific gingipain, Kgp, appears to be the major virulence factor of this organism and here we describe its structure and function. We also discuss the inhibitors of the enzyme produced to date and the potential pathways to newer versions of such molecules that will be required to combat periodontitis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4419-8414-2_2DOI Listing
September 2011

Nerve growth factor inhibits metalloproteinase-disintegrins and blocks ectodomain shedding of platelet glycoprotein VI.

J Biol Chem 2010 Apr 17;285(16):11793-9. Epub 2010 Feb 17.

Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria 3004, Australia.

Nerve growth factor (NGF) plays an important role in regulating mammalian neuronal/embryonic development, angiogenesis, and other physiological processes and has recently been investigated as a potential treatment for the neurodegenerative disorder, Alzheimer disease. In this study, we provide evidence that human NGF may also function as a metalloproteinase inhibitor, based on studies of NGF from snake venom. Originally, our aim was to isolate snake venom metalloproteinases targeting platelet receptors and/or ligands relevant to hemostasis and thrombosis, using Ni(2+)-agarose as a purification step based on the conserved metal ion-coordination motif in venom metalloproteinases. However, subsequent analysis of cobra (Naja kaouthia) venom led to the unexpected discovery that cobra venom NGF bound to Ni(2+)-agarose, eluting at approximately 15 mm imidazole, enabling a one-step purification. The identity of the purified protein was confirmed by mass spectrometry and N-terminal sequence analysis. Partial co-purification of NGF within metalloproteinase-enriched venom fractions led us to test whether NGF affected metalloproteinase activity. Venom NGF potently inhibited metalloproteinases isolated from the same or different venom and specifically bound to purified Nk metalloproteinase immobilized on agarose beads. Human NGF also interacted with human metalloproteinases because it blocked metalloproteinase-mediated shedding of the platelet collagen receptor, glycoprotein (GP)VI, and associated with recombinant ADAM10 by surface plasmon resonance. Together, these results suggest that NGF can function as a metalloproteinase inhibitor.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M110.100479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2852915PMC
April 2010

Characterization of a serine protease homologous to house dust mite group 3 allergens from the scabies mite Sarcoptes scabiei.

J Biol Chem 2009 Dec 7;284(49):34413-22. Epub 2009 Oct 7.

Infectious Diseases and Immunology Division, Queensland Institute for Medical Research, Herston, Queensland 4029, Australia.

The scabies mite, Sarcoptes scabiei var. hominis, infests human skin, causing allergic reactions and facilitating bacterial infection by Streptococcus sp., with serious consequences such as rheumatic fever and rheumatic heart disease. To identify a possible drug target or vaccine candidate protein, we searched for homologues of the group 3 allergen of house dust mites, which we subsequently identified in a cDNA library. The native protein, designated Sar s 3, was shown to be present in the mite gut and excreted in fecal pellets into mite burrows within the upper epidermis. The substrate specificity of proteolytically active recombinant rSar s 3 was elucidated by screening a bacteriophage library. A preference for substrates containing a RS(G/A) sequence at the P1-P2' positions was revealed. A series of peptides synthesized as internally quenched fluorescent substrates validated the phage display data and high performance liquid chromatography/mass spectrometry analysis of the preferred cleaved substrate and confirmed the predicted cleavage site. Searches of the human proteome using sequence data from the phage display allowed the in silico prediction of putative physiological substrates. Among these were numerous epidermal proteins, with filaggrin being a particularly likely candidate substrate. We showed that recombinant rSar s 3 cleaves human filaggrin in vitro and obtained immunohistological evidence that the filaggrin protein is ingested by the mite. This is the first report elucidating the substrate specificity of Sar s 3 and its potential role in scabies mite biology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M109.061911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2797209PMC
December 2009

The gingipains: scissors and glue of the periodontal pathogen, Porphyromonas gingivalis.

Future Microbiol 2009 May;4(4):471-87

Cooperative Research Centre for Oral Health Sciences & Department of Biochemistry & Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.

The anaerobic bacterium, Porphyromonas gingivalis, is associated with chronic periodontal disease (periodontitis or gum disease). The disease is not only the leading cause of tooth loss in the developed world, but is associated with a number of systemic diseases, such as cardiovascular disease and diabetes. The most potent virulence factors of this bacterium are the gingipains, three cysteine proteases that bind and cleave a wide range of host proteins. This article summarizes current knowledge of the structure and function of the enzymes, with a particular focus on what remains to be elucidated regarding the structure and function of the nonenzymatic adhesin domains of the high-molecular-weight forms of the proteases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2217/fmb.09.18DOI Listing
May 2009
-->