Enrico Di Cera Lab

Research Areas


We are interested in the structure, function, regulation, and engineering of trypsin-like proteases and their zymogen forms. The lab offers a unique combination of expertise in enzyme kinetics, molecular biology, thermodynamics, ligand binding and linkage theory, and X-ray structural biology that is ideal for the training of students and postdoctoral fellows interested in the molecular basis of protein function. Our lab engineers and characterizes all the critical reagents that are essential to the performance of our work.


Our long-standing interest in the clotting protease thrombin (1) has led to the functional and structural elucidation of its interaction with Na+ and the role of this interaction in the recognition of 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 kinetic properties (2, 3).


We have solved the structures of thrombin bound to important physiological targets (4-7). We have also elucidated the structures of several thrombin precursors (8, 9) and revealed key aspects of the mechanism of their activation that is relevant to other clotting proteases like protein C (10).


Our recent structural work has led to the identification of conformational selection as a key property of the trypsin fold, with the collapsed E* and open E forms pre-existing in solution for both the protease and its zymogen form (11). Conformational selection is strongly supported by rapid kinetics of ligand binding (12) and offers a unifying framework to understand activity and regulation in trypsin-like proteases.

The E*-E equilibrium defines new strategies of therapeutic intervention, and we have shown how thrombin can be converted into a potent and safe anticoagulant in vitro and in vivo (13) by exploiting the inherent allosteric nature of the trypsin fold.


Click the image below to view a movie illustrating the transition from the E to E* form of thrombin.







Ongoing projects include:
  1. Structural basis of long-range communication among different domains of thrombin
  2. X-ray structure and mechanism of activation of prothrombin
  3. X-ray structure and mechanism of activation of protein C
  4. Engineering autoactivating zymogens
  5. Engineering thrombin for exclusive activity toward protein C






















References

  1. Di Cera E (2008) Thrombin. Mol Aspects Med 29:203-254.
  2. Di Cera E (2006) A structural perspective on enzymes activated by monovalent cations. J Biol Chem 281:1305-1308.
  3. Page MJ, Di Cera E (2006) Role of Na+ and K+ in enzyme function. Physiol Rev 86:1049-1092.
  4. Bah A, Chen Z, Bush-Pelc LA, Mathews FS, Di Cera E (2007) Crystal structures of murine thrombin in complex with the extracellular fragments of murine protease-activated receptors PAR3 and PAR4. Proc Natl Acad Sci USA 104:11603-11608.
  5. Gandhi PS, Chen Z, Appelbaum E, Zapata F, Di Cera E (2011) Stuctural basis of thrombin-PAR interactions. IUBMB Life 63:375-382.
  6. Gandhi PS, Chen Z, Di Cera E (2010) Crystal structure of thrombin bound to the uncleaved extracellular fragment of PAR1. J Biol Chem 285:15393-15398.
  7. Gandhi PS, Chen Z, Mathews FS, Di Cera E (2008) Structural identification of the pathway of long-range communication in an allosteric enzyme. Proc Natl Acad Sci USA 105:1832-1837.
  8. Chen Z, Pelc LA, Di Cera E (2010) Crystal structure of prethrombin-1. Proc Natl Acad Sci U S A 107:19278-19283.
  9. Pozzi N, et al. (2011) Crystal structures of prethrombin-2 reveal alternative conformations under identical solution conditions and the mechanism of zymogen activation. Biochemistry 50:10195-10202.
  10. Gohara DW, Di Cera E (2011) Allostery in trypsin-like proteases suggests new therapeutic strategies. Trends Biotechnol 29:577-585.
  11. Niu W, et al. (2011) Crystallographic and kinetic evidence of allostery in a trypsin-like protease. Biochemistry 50:6301-6307.
  12. Di Cera E (2011) Thrombin as an anticoagulant. Prog Mol Biol Transl Sci 99:145-184.