Deciphering Conformational Changes Associated with the Maturation of Thrombin Anion Binding Exosite I

Billur, Ramya; Ban, David; Sabo, T. Michael; Maurer, Muriel C.

doi:10.1021/acs.biochem.7b00970

Cited by 3 publications

(56 citation statements)

References 68 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…28 NMR buffer composed of 25 mM H 3 PO 4 , 150 mM NaCl, and 0.2 mM EDTA (pH 6.5) was used to dilute the peptide for future NMR studies. 28…”

Section: Synthetic Peptidesmentioning

confidence: 99%

“…were employed. 28 To minimize serine protease autolysis, a 4:1 complex of PPACK to thrombin was incubated at 37 °C for 30 min. Employing a Vivaspin 2 ultrafiltration unit (5000 Da) from Sartorius (Göttingen, Germany), ProT and PPACK-thrombin samples were exchanged into NMR buffer (pH6.5).…”

Section: D Proton Line Broadening and 2d Transferred Noesy Experimentsmentioning

confidence: 99%

“…Interestingly, ABE I ligands derived from hirudin, DNA/RNA aptamers, and PAR3 already have the ability to interact with immature pro-ABE I on ProT. [25][26][27][28] Recent published data with PAR3 (44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56) based peptide ligands demonstrated that solution NMR methods could be used to detect conformational environments that accumulate during exosite maturation. 28 Using individual 15 N-labeled peptides of PAR3G (44-56, P51G), both electrostatic (E48, D54) and hydrophobic PAR3 residues (F47, L52) made unique contributions toward interacting with pro-ABE I and ABE I.…”

Section: Introductionmentioning

confidence: 99%

“…28 Using individual 15 N-labeled peptides of PAR3G (44-56, P51G), both electrostatic (E48, D54) and hydrophobic PAR3 residues (F47, L52) made unique contributions toward interacting with pro-ABE I and ABE I. 28 PAR3 is just one member of the protease activated receptor family. PARs are membrane bound proteins containing seven transmembrane domains, 29 and thrombin exhibits the greatest substrate specificity towards PAR1 30 .…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Thrombin Exosite Maturation and Ligand Binding at ABE II Help Stabilize PAR-Binding Competent Conformation at ABE I

2019

Self Cite

View full text Add to dashboard Cite

Thrombin, derived from zymogen prothrombin (ProT), is a serine protease involved in procoagulation, anticoagulation, and platelet activation. Thrombin's actions are regulated through Anion Binding Exosites I and II (ABE I and ABE II) that undergo maturation during activation. Mature ABEs can utilize exosite-based communication to fulfill thrombin functions. However, the conformational basis behind such long-range communication and the resultant ligand binding affinities are not well understood. Protease Activated Receptors (PARs), involved in platelet activation and aggregation, are known to target thrombin ABE I. Unexpectedly, PAR3 (44-56) can already bind to pro-ABE I of ProT. Nuclear Magnetic Resonance (NMR) ligand-enzyme titrations were used to characterize how individual PAR1 (49-62) residues interact with pro-ABE I and mature ABE I. 1D proton line broadening studies demonstrated that binding affinities for native PAR1P (49-62, P54) and for the weak binding variant PAR1G (49-62, P54G) increased as ProT was converted to mature thrombin. 1 H, 15 N-HSQC titrations revealed that PAR1G residues K51, E53, F55, D58, and E60 exhibited less affinity to pro-ABE I than comparable residues in PAR3G (44-56, P51G). Individual PAR1G residues then displayed tighter binding upon exosite maturation. Long range communication between thrombin exosites was examined by saturating ABE II with phosphorylated GpIbα (269-282, 3Yp) and monitoring binding of PAR1 and PAR3 peptides to ABE I. Individual PAR residues exhibited increased affinities in this dual ligand environment supporting the presence of inter-exosite allostery. Exosite maturation and beneficial long range allostery are proposed to help stabilize an ABE I conformation that can effectively bind PAR ligands.

show abstract

“…28 NMR buffer composed of 25 mM H 3 PO 4 , 150 mM NaCl, and 0.2 mM EDTA (pH 6.5) was used to dilute the peptide for future NMR studies. 28…”

Section: Synthetic Peptidesmentioning

confidence: 99%

Section: D Proton Line Broadening and 2d Transferred Noesy Experimentsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Thrombin Exosite Maturation and Ligand Binding at ABE II Help Stabilize PAR-Binding Competent Conformation at ABE I

2019

Self Cite

View full text Add to dashboard Cite

show abstract

“…These NMR titrations demonstrate that PAR3 residues document structural rearrangements occurring during exosite maturation that are missed by reported X-ray crystal structures. 50,154 Later, the project was extended to better understand the diversity of PAR ligand binding interactions towards zymogen ProT vs mature thrombin. 1D proton line broadening confirmed that PAR1P (49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62) and weaker binding version PAR1G (49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62) already exhibited line broadening with ProT and such broadening increased when bound to thrombin.…”

Section: Discussionmentioning

confidence: 99%

Biophysical exploration of conformational environments in zymogen prothrombin and blood coagulant thrombin.

Billur¹

Self Cite

View full text Add to dashboard Cite

The serine protease thrombin plays important roles in coagulation, anticoagulation, and platelet activation. Thrombin is initially expressed as the inactive zymogen prothrombin (ProT). The final cleaved and activated form of thrombin has mature anion binding exosites (ABEs) and several regulatory loops. Engagement with these exosites helps define the fate of thrombin as a procoagulant or an anticoagulant. Researchers previously reported that zymogen ProT may already bind exosite ligands. Little is known about conformational changes associated with ProT maturation, resultant ligand binding affinities, individual ligand-protein contacts, and long-range communication between thrombin exosites. Protease Activated Receptors (PARs) play critical roles in controlling platelet activation. Using solution NMR methods, we demonstrated that PAR3 (44-56) and weaker binding version PAR3G (44-56, P51G) can already bind to immature pro-ABE I. 1D and 2D 1 H-15 N HSQC titrations revealed that PAR3G 15 N-E48 and 15 N-D54 both entered into higher affinity, intermediate exchange regimes as ProT was converted into thrombin. The high affinity of PAR3G D54 suggests that the thrombin R77a region is better oriented for vii binding than thrombin R75. PAR3G 15 N-F47 and 15 N-L52 experienced significant changes in chemical shift and thus chemical environment upon ABE I maturation. However, the ProT 30s loop made better contacts with PAR3 than the ProT hydrophobic cluster (F34, L65, and I82). The project was extended to PAR1 (49-62). Both PAR1P and weaker version PAR1G (P54G) bound to ProT and proton NMR line broadening increased with thrombin. 1D and 2D 1 H-15 N HSQC titrations revealed that unlike PAR3G (44-56), PAR1G (49-62) 15 N-K51, E53, F55, D58, and E60 exhibited little interactions with ProT. Affinities increased with mature thrombin ABE I. NMR titrations could probe PAR1 (58 DEEKN 62), a region previously unresolved by X-ray crystallography. Interestingly, the ABE II ligand GpIbα (269-282, 3YP) influenced the NMR binding affinities of PAR1G and PAR3G supporting long-range communication between the ABE II and ABE I exosites. Finally, our studies shifted toward the thrombin active site region. Kinetic assays and 2D tr-NOESY studies provided clues on why Fibrinogen Bβ (5-16) is such a weak thrombin substrate. The Fibrinogen Bβ 10 FFSAR 14 sequence contributes towards hindering binding (Km) and product turnover (kcat).

show abstract

Fragment-Based Discovery of Novel VE-PTP Inhibitors Using Orthogonal Biophysical Techniques

Asano,

Yamanaka,

Ohara

et al. 2023

Biochemistry

View full text Add to dashboard Cite

Tyrosine phosphorylation is an essential post-translational modification that regulates various biological events and is implicated in many diseases including cancer and atherosclerosis. Vascular endothelial protein tyrosine phosphatase (VE-PTP), which plays an important role in vascular homeostasis and angiogenesis, is therefore an attractive drug target for these diseases. However, there are still no drugs targeting PTP including VE-PTP. In this paper, we report the discovery of a novel VE-PTP inhibitor, Cpd-2, by fragment-based screening combining various biophysical techniques. Cpd-2 is the first VE-PTP inhibitor with a weakly acidic structure and high selectivity, unlike known strongly acidic inhibitors. We believe that this compound represents a new possibility for the development of bioavailable VE-PTP inhibitors.

show abstract

Deciphering Conformational Changes Associated with the Maturation of Thrombin Anion Binding Exosite I

Cited by 3 publications

References 68 publications

Thrombin Exosite Maturation and Ligand Binding at ABE II Help Stabilize PAR-Binding Competent Conformation at ABE I

Thrombin Exosite Maturation and Ligand Binding at ABE II Help Stabilize PAR-Binding Competent Conformation at ABE I

Biophysical exploration of conformational environments in zymogen prothrombin and blood coagulant thrombin.

Fragment-Based Discovery of Novel VE-PTP Inhibitors Using Orthogonal Biophysical Techniques

Contact Info

Product

Resources

About