We are interested in the structure, function, regulation, and engineering of trypsin-like proteases and their zymogen forms.
We have been working on thrombin since 1990 and elucidated the functional and structural aspects of its interaction with Na+ and physiological substrates. We have cast this work within the larger context of enzymes activated by monovalent cations for which we have offered a classification based on structural and functional properties.
- We have solved and deposited in the Protein Data Bank the X-ray crystal structures of several thrombin precursors (prothrombin, prethrombin-1, and prethrombin-2) and unraveled the molecular basis of their autoactivation.
- We have uncovered the allosteric E*-E equilibrium as a general property of the trypsin fold from structural biology and rapid kinetics.
- We have re-engineered thrombin for selective activity toward protein C in vitro and in vivo and currently have one thrombin construct in preclinical development as an anti-coagulant and anti-inflammatory agent.
- We have elucidated the kinetic signatures of conformational selection and the general relevance of this mechanism for ligand binding.
We have considerable experience in protein engineering and structure-function approaches. We produce all critical reagents that are essential to the performance of our work and characterize their functional properties in terms of the rigor of ligand binding thermodynamics and kinetics. Over the years, we have made these reagents freely available upon request to dozens of investigators in the US and around the world. The environment in our laboratory offers a unique combination of expertise in enzyme kinetics, thermodynamics, ligand binding theory, X-ray structural biology, and protein engineering that is ideal for the training of students and postdoctoral fellows interested in the molecular basis of protein function.
Ligand Binding and Kinetics
- Thermodynamic theory of site-specific binding processes in biological macromolecules. Di Cera E. (1995, Cambridge University Press, Cambridge, UK). Paperback edition published in 2005.
- Site-specific analysis of mutational effects in proteins. Di Cera E. (1998) Adv Protein Chem 51, 59-119. PMID 9615169, PDF.
- Conformational selection or induced fit? A critical appraisal of the kinetic mechanism. Vogt AD, Di Cera E (2012) Biochemistry 51, 5894-5902. PMCID: PMC3550001. PMID 22775458, PDF.
Thrombin and Monovalent Cation Activated Enzymes
- The Na+ binding site of thrombin. Di Cera E, Guinto ER, Vindigni A, Dang QD, Ayala YM, Wuyi M, Tulinsky A. (1995) J Biol Chem 270, 22089-22092. PMID 7673182, PDF.
- Residue 225 determines the Na+-induced allosteric regulation of catalytic activity in serine proteases. Dang QD, Di Cera E. (1996) Proc Natl Acad Sci USA 93, 10653-10656. PMID 8855234, PDF.
- Role of Na+ and K+ in enzyme function. Page MJ, Di Cera E. (2006) Physiol Rev 86, 1049-1092. PMID 17015484, PDF.
Structural changes induced by Na_ binding to thrombin depicted by the structures of the Na+-free (1SGI, yellow) and Na+-bound (1SG8, cyan) forms (Page & Di Cera, 2006).
- Rational engineering of catalytic activity and specificity in a serine protease. Dang QD, Guinto ER, Di Cera E. (1997) Nat Biotechnol 15, 146-149. PMID 9035139, PDF.
- Rational design of a potent anticoagulant thrombin. Cantwell AM, Di Cera E. (2000) J Biol Chem 275, 39827-39830. PMID 11060281, PDF.
- Antithrombotic thrombin variants. Gruber A, Hanson SR, Di Cera E. US Patent 6,706,512 (issued 03.16.04). Expression of thrombin variants. Di Cera E, Gruber A, Gandhi PS, Pelc LA, Pozzi N, Wood DC. US Patent 8,940,297 (issued 01.27.15).
The E*-E Equilibrium in the Trypsin Fold
- Allostery in trypsin-like proteases suggests new therapeutic strategies. Gohara DW, Di Cera E (2011) Trends Biotechnol 29, 577-585. PMCID: PMC3191250. PMID 21726912, PDF.
- Conformational selection in trypsin-like proteases. Pozzi N, Vogt AD, Gohara DW, Di Cera E (2012) Curr Opin Struct Biol 22, 421-431. PMCID: PMC3423485. PMID 22664096, PDF.
- Kinetic dissection of the pre-existing conformational equilibrium in the trypsin fold. Vogt AD, Chakraborty P, Di Cera E. (2015) J Biol Chem 290, 22435-22445. PMID 26216877, PDF.
X-ray crystal structures of thrombin mutant Y225P in the E* (Protein Data Bank 3S7H), E (Protein Data Bank 3S7K), and E:L (Protein Data Bank 1THP) conformations (Vogt 2015).
- Autoactivation of thrombin precursors. Pozzi N, Chen Z, Zapata F, Niu W, Barranco-Medina S, Pelc LA, Di Cera E (2013) J Biol Chem 288, 11601-11610. PMCID: PMC3630838. PMID 23467412, PDF.
- Crystal structure of prothrombin reveals conformational flexibility and mechanism of activation. Pozzi N, Chen Z, Gohara DW, Niu W, Heyduk T, Di Cera E (2013) J Biol Chem 288, 22734-22744. PMCID: PMC3829358. PMID 23775088, PDF.
- The linker connecting the two kringles plays a key role in prothrombin activation. Pozzi N, Chen Z, Pelc LA, Schropshire DB, Di Cera E (2014) Proc Natl Acad Sci USA 111, 7630-7635. PMCID: PMC4040597. PMID 24821807, PDF.
X-ray crystal structure of ProTΔ146–167 in the Ca2+-bound form showing the arrangement of the various domains (Ac, A chain; Bc, B chain; GD, Gla domain; K1, kringle-1; K2, kringle-2) that are not vertically stacked (Pozzi 2014).