A Peptide Bond from the Inter-lobe Segment in the Bilobal Lactoferrin Acts as a Preferred Site for Cleavage for Serine Proteases to Generate the Perfect C-lobe: Structure of the Pepsin Hydrolyzed Lactoferrin C-lobe at 2.28 Å Resolution

2021 ◽  
Author(s):  
Jiya Singh ◽  
Ankit Maurya ◽  
Prashant K. Singh ◽  
V. Viswanathan ◽  
Md Irshad Ahmad ◽  
...  
Keyword(s):  
Serine Proteases ◽  
Peptide Bond

scholarly journals Serine protease dynamics revealed by NMR analysis of the thrombin-thrombomodulin complex

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Riley B. Peacock ◽  
Taylor McGrann ◽  
Marco Tonelli ◽  
Elizabeth A. Komives
Keyword(s):  
Active Site ◽  
Serine Proteases ◽  
Protein C ◽  
Catalytic Mechanism ◽  
Bond Cleavage ◽  
Peptide Bond ◽  
Peptide Bond Cleavage ◽  
Exchange Mass Spectrometry ◽  
Catalytically Active ◽  
Hydrogen Deuterium

AbstractSerine proteases catalyze a multi-step covalent catalytic mechanism of peptide bond cleavage. It has long been assumed that serine proteases including thrombin carry-out catalysis without significant conformational rearrangement of their stable two-β-barrel structure. We present nuclear magnetic resonance (NMR) and hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments on the thrombin-thrombomodulin (TM) complex. Thrombin promotes procoagulative fibrinogen cleavage when fibrinogen engages both the anion binding exosite 1 (ABE1) and the active site. It is thought that TM promotes cleavage of protein C by engaging ABE1 in a similar manner as fibrinogen. Thus, the thrombin-TM complex may represent the catalytically active, ABE1-engaged thrombin. Compared to apo- and active site inhibited-thrombin, we show that thrombin-TM has reduced μs-ms dynamics in the substrate binding (S1) pocket consistent with its known acceleration of protein C binding. Thrombin-TM has increased μs-ms dynamics in a β-strand connecting the TM binding site to the catalytic aspartate. Finally, thrombin-TM had doublet peaks indicative of dynamics that are slow on the NMR timescale in residues along the interface between the two β-barrels. Such dynamics may be responsible for facilitating the N-terminal product release and water molecule entry that are required for hydrolysis of the acyl-enzyme intermediate.

scholarly journals Reversed Proteolysis—Proteases as Peptide Ligases

Catalysts ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 33
Author(s):  
Peter Goettig
Keyword(s):  
Serine Proteases ◽  
Academic Research ◽  
Building Blocks ◽  
Peptide Bond ◽  
Cysteine Proteases ◽  
Small Peptides ◽  
Natural Peptide ◽  
Peptide Esters ◽  
Natural Amino Acids

Historically, ligase activity by proteases was theoretically derived due to their catalyst nature, and it was experimentally observed as early as around 1900. Initially, the digestive proteases, such as pepsin, chymotrypsin, and trypsin were employed to perform in vitro syntheses of small peptides. Protease-catalyzed ligation is more efficient than peptide bond hydrolysis in organic solvents, representing control of the thermodynamic equilibrium. Peptide esters readily form acyl intermediates with serine and cysteine proteases, followed by peptide bond synthesis at the N-terminus of another residue. This type of reaction is under kinetic control, favoring aminolysis over hydrolysis. Although only a few natural peptide ligases are known, such as ubiquitin ligases, sortases, and legumains, the principle of proteases as general catalysts could be adapted to engineer some proteases accordingly. In particular, the serine proteases subtilisin and trypsin were converted to efficient ligases, which are known as subtiligase and trypsiligase. Together with sortases and legumains, they turned out to be very useful in linking peptides and proteins with a great variety of molecules, including biomarkers, sugars or building blocks with non-natural amino acids. Thus, these engineered enzymes are a promising branch for academic research and for pharmaceutical progress.

scholarly journals Inhibition of Staphylococcus aureus cysteine proteases by human serpin potentially limits staphylococcal virulence

2011 ◽  
Vol 392 (5) ◽  
Author(s):  
Tomasz Kantyka ◽  
Karolina Plaza ◽  
Joanna Koziel ◽  
Danuta Florczyk ◽  
Hennig R. Stennicke ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Serine Proteases ◽  
Peptide Bond ◽  
Cysteine Proteases ◽  
Reactive Site ◽  
Association Rate ◽  
High Association ◽  
Endogenous Protease ◽  
Covalent Complex

AbstractBacterial proteases are considered virulence factors and it is presumed that by abrogating their activity, host endogenous protease inhibitors play a role in host defense against invading pathogens. Here we present data showing thatStaphylococcus aureuscysteine proteases (staphopains) are efficiently inhibited by Squamous Cell Carcinoma Antigen 1 (SCCA1), an epithelial-derived serpin. The high association rate constant (kass) for inhibitory complex formation (1.9×104m/s and 5.8×104 m/s for staphopain A and staphopain B interaction with SCCA1, respectively), strongly suggests that SCCA1 can regulate staphopain activityin vivoat epithelial surfaces infected/colonized byS. aureus. The mechanism of staphopain inhibition by SCCA1 is apparently the same for serpin interaction with target serine proteases whereby the formation of a covalent complex result in cleavage of the inhibitory reactive site peptide bond and associated release of the C-terminal serpin fragment. Interestingly, the SCCA1 reactive site closely resembles a motif in the reactive site loop of nativeS. aureus-derived inhibitors of the staphopains (staphostatins). Given thatS. aureusis a major pathogen of epithelial surfaces, we suggest that SCCA1 functions to temper the virulence of this bacterium by inhibiting the staphopains.

scholarly journals Elafin is specifically inactivated by RgpB from Porphyromonas gingivalis by distinct proteolytic cleavage

2009 ◽  
Vol 390 (12) ◽  
Author(s):  
Tomasz Kantyka ◽  
Ties Latendorf ◽  
Oliver Wiedow ◽  
Joachim Bartels ◽  
Regine Gläser ◽  
...  
Keyword(s):  
Porphyromonas Gingivalis ◽  
Serine Proteases ◽  
Peptide Bond ◽  
Cysteine Proteases ◽  
Gingival Tissue ◽  
Proteinase 3 ◽  
Host Proteins ◽  
Surface Loop ◽  
Endogenous Proteins ◽  
Causative Bacterium

AbstractPorphyromonas gingivalis, the major causative bacterium of periodontitis, contributes significantly to elevated proteolytic activity at periodontal pockets owing to the presence of both bacteria and host, predominantly neutrophil-derived, serine proteases. Normally the activity of the latter enzymes is tightly regulated by endogenous proteins, including elafin, a potent neutrophil elastase and proteinase 3 inhibitor released from epithelial cells at sites of inflammation. Here, we report that all three gingipains (HRgpA, RgpB, and Kgp) have the ability to degrade elafin, with RgpB being far more efficient than other gingipains. RgpB efficiently inactivates the inhibitory activity of elafin at subnanomolar concentrations through proteolysis limited to the Arg22-Cys23 peptide bond within the surface loop harboring the inhibitor active site. Notably, elafin resists inactivation by severalStaphylococcus aureus-derived serine and cysteine proteases, confirming the high stability of this protein against proteolytic degradation. Therefore, we conclude that elafin inactivation by RgpB represents a specific pathogenic adaptation ofP. gingivalisto disturb the protease-protease inhibitor balance in the infected gingival tissue. This contributes to enhanced degradation of host proteins and generation of a pool of peptides serving as nutrients for this asaccharolytic pathogen.

scholarly journals Functional study of elafin cleaved by Pseudomonas aeruginosa metalloproteinases

2010 ◽  
Vol 391 (6) ◽  
Author(s):  
Nicolas Guyot ◽  
Gudmundur Bergsson ◽  
Marcus W. Butler ◽  
Catherine M. Greene ◽  
Sinéad Weldon ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Serine Proteases ◽  
Antibacterial Properties ◽  
Peptide Bond ◽  
Matrix Proteins ◽  
Functional Study ◽  
Pulmonary Epithelium ◽  
Amino Terminal ◽  
Alternative Sites ◽  
Binding Loop

AbstractElafin is a 6-kDa innate immune protein present at several epithelial surfaces including the pulmonary epithelium. It is a canonical protease inhibitor of two neutrophil serine proteases [neutrophil elastase (NE) and proteinase 3] with the capacity to covalently bind extracellular matrix proteins by transglutamination. In addition to these properties, elafin also possesses antimicrobial and immunomodulatory activities. The aim of the present study was to investigate the effect ofPseudomonas aeruginosaproteases on elafin function. We found thatP. aeruginosaPAO1-conditioned medium and two purifiedPseudomonasmetalloproteases, pseudolysin (elastase) and aeruginolysin (alkaline protease), are able to cleave recombinant elafin. Pseudolysin was shown to inactivate the anti-NE activity of elafin by cleaving its protease-binding loop. Interestingly, antibacterial properties of elafin against PAO1 were found to be unaffected after pseudolysin treatment. In contrast to pseudolysin, aeruginolysin failed to inactivate the inhibitory properties of elafin against NE. Aeruginolysin cleaves elafin at the amino-terminal Lys6-Gly7 peptide bond, resulting in a decreased ability to covalently bind purified fibronectin following transglutaminase activity. In conclusion, this study provides evidence that elafin is susceptible to proteolytic cleavage at alternative sites byP. aeruginosametalloproteinases, which can affect different biological functions of elafin.

scholarly journals SepM, a Streptococcal Protease Involved in Quorum Sensing, Displays Strict Substrate Specificity

2015 ◽  
Vol 198 (3) ◽  
pp. 436-447 ◽  
Author(s):  
Saswati Biswas ◽  
Luyang Cao ◽  
Albert Kim ◽  
Indranil Biswas
Keyword(s):  
Quorum Sensing ◽  
Substrate Specificity ◽  
Serine Proteases ◽  
Signal Transduction Pathway ◽  
Peptide Bond ◽  
Leader Peptide ◽  
Small Peptides ◽  
Generate Functional ◽  
Content Type ◽  
Two Component Signal Transduction

ABSTRACTStreptococcus mutans, a causative agent of dental caries, relies on multiple quorum-sensing (QS) pathways that coordinate the expression of factors needed for colonization in the oral cavity.S. mutansuses small peptides as QS signaling molecules that typically are secreted into the outside milieu. Competence-stimulating peptide (CSP) is one such QS signaling molecule that functions through the ComDE two-component signal transduction pathway. CSP is secreted through NlmTE, a dedicated ABC transporter that cleaves off the N-terminal leader peptide to generate a mature peptide that is 21 residues long (CSP-21). We recently identified a surface-localized protease, SepM, which further cleaves the CSP-21 peptide at the C-terminal end and removes the last 3 residues to generate CSP-18. CSP-18 is the active QS molecule that interacts with the ComD sensor kinase to activate the QS pathway. In this study, we show that SepM specifically cleaves CSP-21 between the Ala18 and Leu19 residues. We also show that SepM recognizes only Ala at position 18 and Leu at position 19, although some CSP-18 variants with a substitution at position 18 can function equally as well as the QS peptide. Furthermore, we demonstrate that SepM homologs from other streptococci are capable of processing CSP-21 to generate functional CSP-18.IMPORTANCESepM is a membrane-associated streptococcal protease that processes competence-stimulating peptide (CSP) to generate an active quorum-sensing molecule inS. mutans. SepM belongs to the S16 family of serine proteases, and in this study, we found that SepM behaves as an endopeptidase. SepM displays strict substrate specificity and cleaves the peptide bond between the Ala and Leu residues. This is the first report of an endopeptidase that specifically cleaves these two residues.

Structural and Functional Significance of Amino Acid Lysine192 (chymotrypsin numbering) in Factor XIa and Factor VIIa.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2030-2030
Author(s):  
Amy E Schmidt ◽  
Sayeh Agah ◽  
Mao-fu Sun ◽  
Kaillathe Padmanabhan ◽  
Duilio Cascio ◽  
...  
Keyword(s):  
Amino Acid ◽  
Serine Proteases ◽  
Peptide Bond ◽  
Side Chain ◽  
Hole Formation ◽  
Oxyanion Hole ◽  
Structural Role ◽  
Inhibitor Specificity ◽  
Modeling Studies ◽  
Substrate Inhibitor

Abstract In blood coagulation serine proteases, amino acid 192 (chymotrypsin numbering) influences substrate and inhibitor specificity, and also forms a peptide bond with Gly193, a critical part of the protease oxyanion hole. Residue 192 is either Glu or Gln in several serine proteases including thrombin, factor (F) Xa, FIXa, and activated protein C. However, it is Lys in factor FXIa and FVIIa. Both FXIa and FVIIa/tissue factor (TF) share FIX as their substrate. In addition, FVIIa/TF activates FX and is inhibited by TF pathway inhibitor (TFPI), whereas FXIa is inhibited by diisopropylfluorophosphate (DFP), p-aminobenzamidine (p-AB), antithrombin and amyloid-b precursor protein Kunitz domain (AbPP). We investigated the importance of Lys192 in FXIa and FVIIa for substrate/inhibitor specificity and oxyanion hole formation. Recombinant FXIa was prepared with Pro (FXIaK192P), Glu (FXIaK192E) or Gln (FXIaK192Q) substitutions for Lys192. Rate constants for inhibition by DFP were similar for wild type FXIa (FXIaWT), FXIaK192E and FXIaK192Q, but 80-fold slower for FXIaK192P. Only FXIaK192P differed in binding to pAB compared to FXIaWT. These data indicate that Pro192 causes distortion of the oxyanion hole and S1 binding site. All mutants bound the FXIa substrate FIX normally. However, catalysis of FIX was impaired 400, 35, and 110-fold for FXIaK192P, FXIaK192E, and FXIaK192Q, respectively, while k2 for inhibition by antithrombin was reduced 160, 15 and 16-fold. FXIaK192P bound to AbPP with 800-fold decreased affinity; while FXIaK192E and FXIaK192Q bound with 6-10-fold higher affinity. Modeling studies indicate that there is loss of the normal interaction between Lys192 and the substrate/inhibitor P3’ residue in interactions between FXIaK192E and FXIaK192Q and factor IX and antithrombin. The Ki for Glu and Gln mutant inhibition by AbPP is slightly better probably due to the ability of these two amino acids to make two H-bonds as compared to only one for Lys192. Lys192 makes H-bond with the carbonyl O of Gly12 in AbPP, whereas Glu and Gln at this position make H-bonds with the carbonyl O of Cys14 and Ala16 in AbPP. In FXIaK192P, the oxyanion hole is impaired due to a flip in the 192-193 peptide bond causing the amide N of Gly193 to point away from the oxyanion hole, resulting in a severe impairment of all catalytic functions. Thus a non-proline residue 192 is essential for proper oxyanion hole formation in serine proteases, and Lys192 in FXIa contributes significantly towards macromolecular substrate catalysis and inhibitor binding. In contrast to FXIaWT, the 192-193 peptide bond in FVIIaWT ± TF is already in a nonstandard conformation in which the amide N of Gly193 points away, and the carbonyl O of the Lys192 points into the oxyanion hole (Bajaj, et al., J. Biol. Chem.281, 24873-24888, 2006). The H-bond between the amide N of Gly193 and the carbonyl side chain of Gln143, a residue unique to FVIIa, maintains this nonstandard conformation in FVIIa. Notably, upon substrate binding the 192-193 bond flips 180° such that the oxyanion hole is fully formed. Now the H-bond is formed between the amide N of Gly193 and the carbonyl oxygen of the transition state intermediate. For this to occur, the H-bond between the amide N of Gly193 and the carbonyl side chain of Gln143 must first be broken. Previous studies have shown that changing Lys192 to Glu or Gln in FVIIa severely impairs its biologic activity. However, the structural basis for this impairment is not understood. We performed molecular modeling studies which reveal that the side chain of the Glu192 carboxyl group or of the Gln192 carbonyl group makes strong H-bond with the side chain NH2 group of Gln143; this is not possible for Lys192. Thus in Glu192 or Gln192 mutant of FVIIa, it is energetically difficult to break the H-bond between the amide N of Gly193 and the carbonyl side chain of Gln143 because the H-bond involving the side chain of Glu192 or Gln192 and the Gln143 must be simultaneously broken. This provides a structural rational for the impaired function of FVIIaK192E or FVIIaK192Q. To test this concept further, we prepared FVIIa in which Lys192 was replaced with alanine. All properties of this alanine mutant were normal substantiating the proposed structural role of Lys192 in FVIIa. This structural role is unique to FVIIa among all known serine proteases, as is the presence of residue Gln143 that contributes to this phenomenon.

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