TAFIa inhibiting nanobodies as profibrinolytic tools and discovery of a new TAFIa conformation

Hendrickx, Maarten; Winter, Alan D.; Buelens, K.; Compernolle, Griet; Hassanzadeh-Ghassabeh, Gholamreza; Gils, Ann; Declerck, Paul

doi:10.1111/j.1538-7836.2011.04495.x

Cited by 24 publications

(26 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Anti-muPA nanobodies were identified by a polyclonal phage ELISA by randomly picking single colonies. Positive clones were sequenced and unique clones were transformed into E. coli WK6 (su − ) cells and produced as described previously 34 .…”

Section: Methodsmentioning

confidence: 99%

Discovery of a novel conformational equilibrium in urokinase-type plasminogen activator

Kromann-Hansen

Lange

Sørensen

et al. 2017

Sci Rep

View full text Add to dashboard Cite

Although trypsin-like serine proteases have flexible surface-exposed loops and are known to adopt higher and lower activity conformations, structural determinants for the different conformations have remained largely obscure. The trypsin-like serine protease, urokinase-type plasminogen activator (uPA), is central in tissue remodeling processes and also strongly implicated in tumor metastasis. We solved five X-ray crystal structures of murine uPA (muPA) in the absence and presence of allosteric molecules and/or substrate-like molecules. The structure of unbound muPA revealed an unsuspected non-chymotrypsin-like protease conformation in which two β-strands in the core of the protease domain undergoes a major antiparallel-to-parallel conformational transition. We next isolated two anti-muPA nanobodies; an active-site binding nanobody and an allosteric nanobody. Crystal structures of the muPA:nanobody complexes and hydrogen-deuterium exchange mass spectrometry revealed molecular insights about molecular factors controlling the antiparallel-to-parallel equilibrium in muPA. Together with muPA activity assays, the data provide valuable insights into regulatory mechanisms and conformational flexibility of uPA and trypsin-like serine proteases in general.

show abstract

Section: Methodsmentioning

confidence: 99%

Discovery of a novel conformational equilibrium in urokinase-type plasminogen activator

Kromann-Hansen

Lange

Sørensen

et al. 2017

Sci Rep

View full text Add to dashboard Cite

show abstract

“…uPA-binding nanobodies were identified by a polyclonal phage ELISA by randomly picking single colonies. Positive clones were sequenced, and unique clones were transformed into E. coli WK6 (su Ϫ ) cells, produced and purified as described previously (39). Alanine mutants of Nb4 were prepared by standard QuickChange site-directed mutagenesis and expressed in E. coli WK6 (su Ϫ ) cells.…”

Section: Methodsmentioning

confidence: 99%

A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior

Kromann-Hansen

Oldenburg

Yung

et al. 2016

Journal of Biological Chemistry

View full text Add to dashboard Cite

A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30 -40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors.Serine proteases catalyze the hydrolysis of peptide bonds and are involved in numerous physiological processes, including digestion, blood clotting, fibrinolysis, complement activation, and turnover of the extracellular matrix (1). Neutralizing serine protease activity using orthosteric inhibitors, i.e. active site binding inhibitors, has been shown to be a successful therapeutic strategy for a number of pathological conditions, although the similar active site topology in all serine proteases increases the risk of off-target effects. Today, serine protease inhibitors are clinically used for therapy of several diseases, including thrombosis and bleeding disorders (2-4).All serine proteases catalyze the same type of hydrolytic reaction utilizing the same biochemical mechanism. Serine protease-catalyzed hydrolysis of a scissile bond proceeds through a highly conserved mechanism involving two tetrahedral intermediates and an acyl-enzyme complex. The polypeptide substrate is aligned in the active site of the protease interacting with the substrate specificity pockets denoted S1-Sn and S1Ј-SnЈ on the acyl and leaving group side of the scissile bond, respectively (5). The P1 residue of the substrate binds into the S1 pocket, and its carbonyl oxygen atom is inserted into the so-called oxyanion hole (backbone amides of chymotrypsinogen numbering). The catalytic triad (His-57, in the protease generates the required nucleophile for the attack of the hydroxyl group of Ser-195 on the carbonyl group of the P1-P1Ј scissile bond to form the first tetrahedral intermediate and later the acyl-enzyme. Following release of the P1Ј-leaving group, a water molecule performs a second nucleophilic attack, thereby completing the cycle (6).Peptide segments that bind the active site of serine proteases in a substrate-like manner may behave like an inhibitor or substrate. Ho...

show abstract

“…The preference of VHH paratopes to interact with cavities on the antigen enables Nanobodies against enzymes to modulate the catalytic activity of the antigen, as the substrate recognition site of enzymes is located within concave epitopes [27–29]. For these reasons, Nanobodies find applications as enzyme inhibitors, or as research tools to unravel an enzymatic mechanism [27,29–31].…”

Section: Camels and Llamasmentioning

confidence: 99%

“…For these reasons, Nanobodies find applications as enzyme inhibitors, or as research tools to unravel an enzymatic mechanism [27,29–31]. Likewise, the interaction between viral ligands and host receptors is also mediated by concave to convex surfaces.…”

Section: Camels and Llamasmentioning

confidence: 99%

Distinct antibody species: structural differences creating therapeutic opportunities

Muyldermans

Smider

2016

Current Opinion in Immunology

View full text Add to dashboard Cite

Antibodies have been a remarkably successful class of molecules for binding a large number of antigens in therapeutic, diagnostic, and research applications. Typical antibodies derived from mouse or human sources use the surface formed by complementarity determining regions (CDRs) on the variable regions of the heavy chain/light chain heterodimer, which typically forms a relatively flat binding surface. Alternative species, particularly camelids and bovines, provide a unique paradigm for antigen recognition through novel domains which form the antigen binding paratope. For camelids, heavy chain antibodies bind antigen with only a single heavy chain variable region, in the absence of light chains. In bovines, ultralong CDR-H3 regions form an independently folding minidomain, which protrudes from the surface of the antibody and is diverse in both its sequence and disulfide patterns. The atypical paratopes of camelids and bovines potentially provide the ability to interact with different epitopes, particularly recessed or concave surfaces, compared to traditional antibodies.

show abstract

TAFIa inhibiting nanobodies as profibrinolytic tools and discovery of a new TAFIa conformation

Cited by 24 publications

References 32 publications

Discovery of a novel conformational equilibrium in urokinase-type plasminogen activator

Discovery of a novel conformational equilibrium in urokinase-type plasminogen activator

A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior

Distinct antibody species: structural differences creating therapeutic opportunities

Contact Info

Product

Resources

About