Structural and mechanistic insight into how antibodies inhibit serine proteases

2010 ◽  
Vol 430 (2) ◽  
pp. 179-189 ◽  
Author(s):  
Rajkumar Ganesan ◽  
Charles Eigenbrot ◽  
Daniel Kirchhofer
Keyword(s):  
Active Site ◽  
Serine Proteases ◽  
Structural Changes ◽  
Competitive Inhibition ◽  
Substrate Binding ◽  
Structural Features ◽  
Inhibition Mechanism ◽  
Interaction Sites ◽  
Binding Cleft ◽  
Insight Into

Antibodies display great versatility in protein interactions and have become important therapeutic agents for a variety of human diseases. Their ability to discriminate between highly conserved sequences could be of great use for therapeutic approaches that target proteases, for which structural features are conserved among family members. Recent crystal structures of antibody–protease complexes provide exciting insight into the variety of ways antibodies can interfere with the catalytic machinery of serine proteases. The studies revealed the molecular details of two fundamental mechanisms by which antibodies inhibit catalysis of trypsin-like serine proteases, exemplified by hepatocyte growth factor activator and MT-SP1 (matriptase). Enzyme kinetics defines both mechanisms as competitive inhibition systems, yet, on the molecular level, they involve distinct structural elements of the active-site region. In the steric hindrance mechanism, the antibody binds to protruding surface loops and inserts one or two CDR (complementarity-determining region) loops into the enzyme's substrate-binding cleft, which results in obstruction of substrate access. In the allosteric inhibition mechanism the antibody binds outside the active site at the periphery of the substrate-binding cleft and, mediated through a conformational change of a surface loop, imposes structural changes at important substrate interaction sites resulting in impaired catalysis. At the centre of this allosteric mechanism is the 99-loop, which is sandwiched between the substrate and the antibody-binding sites and serves as a mobile conduit between these sites. These findings provide comprehensive structural and functional insight into the molecular versatility of antibodies for interfering with the catalytic machinery of proteases.

scholarly journals Mitochondrial ClpX activates an essential biosynthetic enzyme through partial unfolding

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Julia R Kardon ◽  
Jamie A Moroco ◽  
John R Engen ◽  
Tania A Baker
Keyword(s):  
Active Site ◽  
Autonomous System ◽  
Aminolevulinic Acid ◽  
Structural Features ◽  
Biosynthetic Enzyme ◽  
Cofactor Binding ◽  
Partial Unfolding ◽  
Chaperone Interactions ◽  
Aaa Protein ◽  
Insight Into

Mitochondria control the activity, quality, and lifetime of their proteins with an autonomous system of chaperones, but the signals that direct substrate-chaperone interactions and outcomes are poorly understood. We previously discovered that the mitochondrial AAA+ protein unfoldase ClpX (mtClpX) activates the initiating enzyme for heme biosynthesis, 5-aminolevulinic acid synthase (ALAS), by promoting cofactor incorporation. Here, we ask how mtClpX accomplishes this activation. Using S. cerevisiae proteins, we identified sequence and structural features within ALAS that position mtClpX and provide it with a grip for acting on ALAS. Observation of ALAS undergoing remodeling by mtClpX revealed that unfolding is limited to a region extending from the mtClpX-binding site to the active site. Unfolding along this path is required for mtClpX to gate cofactor binding to ALAS. This targeted unfolding contrasts with the global unfolding canonically executed by ClpX homologs and provides insight into how substrate-chaperone interactions direct the outcome of remodeling.

scholarly journals Purification and characterization of Ak.1 protease, a thermostable subtilisin with a disulphide bond in the substrate-binding cleft

2000 ◽  
Vol 350 (1) ◽  
pp. 321-328 ◽  
Author(s):  
Helen S. TOOGOOD ◽  
Clyde A. SMITH ◽  
Edward N. BAKER ◽  
Roy M. DANIEL
Keyword(s):  
Active Site ◽  
Substrate Binding ◽  
Fold Increase ◽  
Branched Chain Amino Acids ◽  
Disulphide Bond ◽  
Purification And Characterization ◽  
Active Site Cleft ◽  
Binding Cleft ◽  
Branched Chain

Ak.1 protease, a thermostable subtilisin isolated originally from Bacillus st. Ak.1, was purified to homogeneity from the Escherichia coli clone PB5517. It is active against substrates containing neutral or hydrophobic branched-chain amino acids at the P1 site, such as valine, alanine or phenylalanine. The Km and kcat of the enzyme decrease with decreasing temperature, though not to the same degree with all substrates, suggesting that specificity changes with temperature. The protease is markedly stabilized by Ca2+ ions. At 70°C, a 10-fold increase in Ca2+ concentration increases the half-life by three orders of magnitude. Ak.1 protease is stabilized by Ca2+ to a greater extent than is thermitase. This may be due, in part, to the presence of an extra Ca2+-binding site in Ak.1 protease. Other metal ions, such as Sr2+, increase the thermostability of the enzyme, but to a significantly lower degree than does Ca2+. The structure of the protease showed the presence of a disulphide bond located within the active-site cleft. This bond influences both enzyme activity and thermostability. The disulphide bond appears to have a dual role: maintaining the integrity of the substrate-binding cleft and increasing the thermostability of the protease. The protease was originally investigated to determine its usefulness in the clean-up of DNA at high temperatures. However, it was found that this protease has a limited substrate specificity, so this application was not explored further.

scholarly journals Structural features of ring C of 20-oxo steroids and the interaction with cortisone reductase

1974 ◽  
Vol 137 (2) ◽  
pp. 349-354 ◽  
Author(s):  
Ian H. White ◽  
Jonathan Jeffery
Keyword(s):  
Structural Changes ◽  
Substrate Binding ◽  
Structural Features ◽  
Hydrophobic Region ◽  
Kinetic Measurements ◽  
Polar Substituent ◽  
Km Value ◽  
Nad Oxidoreductase ◽  
Polar Interactions ◽  
Electronic Character

Kinetic measurements were made with cortisone reductase (20-dihydrocortisone–NAD+ oxidoreductase, EC 1.1.1.53) and a series of substrates which differed in shape, size and electronic character in the region adjacent to C-11, C-14 and C-18. Structural changes at C-11 in these substrates resulted in up to 660-fold changes in the apparent Km value, up to 200-fold changes in the apparent Vmax. value and up to 800-fold changes in the ratio of these kinetic constants. It is suggested that interactions important for substrate function normally occur between the enzyme and the C ring in the region of C-11, that these interactions arise from so-called hydrophobic forces between the generally hydrophobic C ring portion of the substrate and a hydrophobic region of the enzyme, but that when the substrate contains a polar substituent in this portion of the molecule, then polar interactions with polar moieties of the enzyme can also be important. It is further suggested that the part of the enzyme that interacts with the region of C-11 in the substrate is flexible, and that substrate binding involves at least some degree of induced fit.

scholarly journals Inhibition of enzyme activity of and cell-mediated substrate cleavage by membrane type 1 matrix metalloproteinase by newly developed mercaptosulphide inhibitors

2005 ◽  
Vol 392 (3) ◽  
pp. 527-536 ◽  
Author(s):  
Douglas R. Hurst ◽  
Martin A. Schwartz ◽  
Yonghao Jin ◽  
Mohammad A. Ghaffari ◽  
Pallavi Kozarekar ◽  
...  
Keyword(s):  
Matrix Metalloproteinase ◽  
Active Site ◽  
Competitive Inhibition ◽  
Human Cancer ◽  
Calf Serum ◽  
Inhibition Mechanism ◽  
Air Oxidation ◽  
Substrate Cleavage ◽  
Membrane Type

MT1-MMP (membrane type 1 matrix metalloproteinase, or MMP-14) is a key enzyme in molecular carcinogenesis, tumour-cell growth, invasion and angiogenesis. Novel and potent MMP inhibitors with a mercaptosulphide zinc-binding functionality have been designed and synthesized, and tested against human MT1-MMP and other MMPs. Binding to the MT1-MMP active site was verified by the competitive-inhibition mechanism and stereochemical requirements. MT1-MMP preferred deep P1′ substituents, such as homophenylalanine instead of phenylalanine. Novel inhibitors with a non-prime phthalimido substituent had Ki values in the low-nanomolar range; the most potent of these inhibitors was tested and found to be stable against air-oxidation in calf serum for at least 2 days. To illustrate the molecular interactions of the inhibitor–enzyme complex, theoretical docking of the inhibitors into the active site of MT1-MMP and molecular minimization of the complex were performed. In addition to maintaining the substrate-specificity pocket (S1′ site) van der Waals interactions, the P1′ position side chain may be critical for the peptide-backbone hydrogen-bonding network. To test the inhibition of cell-mediated substrate cleavage, two human cancer-cell culture models were used. Two of the most potent inhibitors tested reached the target enzyme and effectively inhibited activation of proMMP-2 by endogenous MT1-MMP produced by HT1080 human fibrosarcoma cells, and blocked fibronectin degradation by prostate cancer LNCaP cells stably transfected with MT1-MMP. These results provide a model for mercaptosulphide inhibitor binding to MT1-MMP that may aid in the design of more potent and selective inhibitors for MT1-MMP.

Papain-like lysosomal cysteine proteases and their inhibitors: drug discovery targets?

2003 ◽  
Vol 70 ◽  
pp. 15-30 ◽  
Author(s):  
Dŭsan Turk ◽  
Boris Turk ◽  
Vito Turk
Keyword(s):  
Drug Discovery ◽  
Active Site ◽  
Binding Sites ◽  
Substrate Binding ◽  
Cysteine Proteases ◽  
Structural Features ◽  
Discovery Research ◽  
Drug Discovery Research ◽  
Lysosomal Cysteine Proteases ◽  
Substrate Binding Sites

Papain-like lysosomal cysteine proteases are processive and digestive enzymes that are expressed in organisms from bacteria to humans. Increasing knowledge about the physiological and pathological roles of cysteine proteases is bringing them into the focus of drug discovery research. These proteases have rather short active-site clefts, comprising three well defined substrate-binding subsites (S2, S1 and S1') and additional broad binding areas (S4, S3, S2' and S3'). The geometry of the active site distinguishes cysteine proteases from other protease classes, such as serine and aspartic proteases, which have six and eight substrate-binding sites respectively. Exopeptidases (cathepsins B, C, H and X), in contrast with endopeptidases (such as cathepsins L, S, V and F), possess structural features that facilitate the binding of N- and C-terminal groups of substrates into the active-site cleft. Other than a clear preference for free chain termini in the case of exopeptidases, the substrate-binding sites exhibit no strict specificities. Instead, their subsite preferences arise more from the specific exclusion of substrate types. This presents a challenge for the design of inhibitors to target a specific cathepsin: only the cumulative effect of an assembly of inhibitor fragments will bring the desired result.

scholarly journals Inhibitors of nicotinamide N-methyltransferase designed to mimic the methylation reaction transition state

2017 ◽  
Vol 15 (31) ◽  
pp. 6656-6667 ◽  
Author(s):  
Matthijs J. van Haren ◽  
Rebecca Taig ◽  
Jilles Kuppens ◽  
Javier Sastre Toraño ◽  
Ed E. Moret ◽  
...  
Keyword(s):  
Transition State ◽  
Active Site ◽  
Substrate Binding ◽  
Structural Features ◽  
Methylation Reaction ◽  
Potent Inhibition ◽  
Binding Pockets

Inhibitors designed to simultaneously occupy the different substrate binding pockets of the NNMT active site reveal key structural features required for potent inhibition.

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