Interplay between conformational selection and zymogen activation

Author: Will Bowman

https://www.nature.com/articles/s41598-018-21728-9

Interplay between conformational selection and zymogen activation

Pradipta Chakraborty, Laura Acquasaliente, Leslie A. Pelc Enrico Di Cera

 

In this paper, authors are exploring the relationship between the conformational state of protease precursors, zymogens, and their relative affinity and catalytic ability, Proteases are a class of proteins that are involved in degradation of amide bonds in other proteins, either completely degrading a protein to recycle its amino acids or for other more specific purposes. Proteases, with their active role of degrading other proteins, are not left to float about in solution active at all times; these proteins are kept in what is canonically known as an “off” state in the form of a zymogen. Zymogens are basically protease precursors, containing residues and domains that must first be removed before the protease can function2.

 

The authors are specifically exploring the protease thrombin, and its zymogen: prothrombin; this trypsin family protease has roles in blood coagulation1. Prothrombin contains a Gla domain, 2 Kringle domains, and the protease domain. Conversion to thrombin is achieved by shedding the excess domains7. Prothrombin can also be converted into prethrombin-1 and 2, as well as meizothrombin

 

Thrombin is generated from prothrombin through the cleavage of R271 and R320, removing the Gla domain and both Kringle domains. Prethrombin-2 is formed by only cleaving at R271. Sole cleavage at R320 generates meizothrombin. Prethrombin-1 is formed by cleavage at R155, only removing the Gla and the first Kringle domain.

 

Previously in the literature, zymogen activation was described solely by the Huber-Bode mechanism2. This mechanism implies that nearly all activity(E) is associated with the protease state, and all inactivity(E*) with the zymogen state. However, this fails to account for the body of literature observing spontaneous zymogen activation3,4 and inactive proteases5. From this, authors are proposing an alternative mechanism: activity is defined on a sliding scale from zymogen to protease, where activity is seen to increase from one to the other, but is not limited to a single form.

 

The trypsin fold is known to be very flexible, as seen in the various configurations of the 215-217 segment seen in PDB crystal structure database6. Authors speculate that this is only a snapshot of the actual conformations present in solution. This region is important as it helps to define the active site and provides anchor points for the substrates. Such a region would be ideal to observe the active site and relate information to the activity of the protein. NMR measurements are usually well suited to such a task of observing the states of this region in solution, but have so far limited.

 

Authors decide to use rapid kinetic experiments to detail the activity of the various forms of zymogen (prothrombin, meizothrombin, prethrombin-1 and 2) and protease (thrombin). Rapid kinetics of ligand binding to protease active sites support a mechanism which allows for optimal conformations to be selected from a pre-existing ensemble.

 

Authors first run rapid kinetics of FPR binding to both meizothrombin and thrombin. In both experiments, the data received is similar; both forms have two relaxations that increase linearly and hyperbolically with ligand concentrations. This kinetic profile is consistent with a scheme

E* (<- k21)(k12 ->) E (<- koff)(kon ->) E:L

Based on the similarities in data, authors conclude that once R320 is cleaved, any remaining auxiliary domain is inconsequential to activity.  

 

Further experimentation showed that in another rapid kinetics experiment with prothrombin, only one linear relaxation with ligand concentration was observed. Authors determine that this result further confirms their reaction scheme. Prothrombin is shown to be about 700 times less active than thrombin. This is explained by the fact that active site opening (k12) is the same is both forms, but closing (k21) is 10 times faster in prothrombin. This is conjunction with prothrombin’s slower dissociation rate.

 

A final set of experiments used prethrombin-1 and 2 in rapid kinetics. Prethrombin-1 had a single relaxation independent of ligand concentrations, which when combined with other data found implies that koff does not change considerably upon removal of Gla and first Kringle domain. However, for prethrombin-1, k12 values did increase, and therefore increased activity over prothrombin. Data from prethrombin-2 shows a single relaxation that increases hyperbolically with ligand concentration, and k12 values increase over prethrombin-1.

 

This data leads authors to believe that as the excess domains are removed, catalytic activity is improved by the organization of the active site.

 

Based on the data found, authors are able to determine that PDB structures are indeed showing evidence for variable trypsin folding patterns that allow for different active site accessibility. This allows for a new way of thinking about how zymogens are activated and why poorly active proteases are not more active. Stabilization of E* states would prevent protease activity, despite its zymogen to protease ensemble have active capability.

 

The authors have illustrated a wonderful point about the complexity of zymogen activation and protease formation. This new work overturns a lot of the canonical ways of thinking about zymogen activity. In the context of a classroom, it is incredibly important not only to know the current acceptable model for protein function, but be able to grapple and understand the complexity that biological systems have. Just as metabolism can be overshadowed by off-target syntheses and onco-metabolites, zymogen activation should be understood as the complex process that it is. The authors have done a wonderful job of illustrating this complex picture.

 

References:

 

  1. Page, M. J. & Di Cera, E. Serine peptidases: classification, structure and function. Cell Mol Life Sci 65, 1220–1236 (2008).
  2. Huber, R. & Bode, W. Structural basis of the activation and action of trypsin. Acc Chem Res11, 114–122 (1978).
  3. Yamamoto, E., Kitano, Y. & Hasumi, K. Elucidation of crucial structures for a catechol-based inhibitor of plasma hyaluronan-binding protein (factor VII activating protease) autoactivation. Biosci Biotechnol Biochem 75, 2070–2 (2011).
  4. Sichler, K. et al. Crystal structures of uninhibited factor VIIa link its cofactor and substrate-assisted activation to specific interactions. J Mol Biol 322, 591–603 (2002).
  5. Edgington, T. S., Mackman, N., Brand, K. & Ruf, W. The structural biology of expression and function of tissue factor. Thromb Haemost 66, 67–79 (1991).
  6. Rohr, K. B. et al. X-ray structures of free and leupeptin-complexed human alphaI-tryptase mutants: indication for an alpha–>beta-tryptase transition. J Mol Biol 357, 195–209 (2006).
  7. Degen, S. J. & Davie, E. W. Nucleotide sequence of the gene for human prothrombin. Biochemistry 26, 6165–77 (1987).

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