Trypsinogen activation as observed in accelerated molecular dynamics simulations

Abstract

Serine proteases are involved in many fundamental physiological processes, and control of their activity mainly results from the fact that they are synthetized in an inactive form that becomes active upon cleavage. Three decades ago Martin Karplus's group performed the first molecular dynamics simulations of trypsin, the most studied member of the serine protease family, to address the transition from the zymogen to its active form. Based on the computational power available at the time, only high frequency fluctuations, but not the transition steps, could be observed. By performing accelerated molecular dynamics (aMD) simulations, an interesting approach that increases the configurational sampling of atomistic simulations, we were able to observe the N-terminal tail insertion, a crucial step of the transition mechanism. Our results also support the hypothesis that the hydrophobic effect is the main force guiding the insertion step, although substantial enthalpic contributions are important in the activation mechanism. As the Nterminal tail insertion is a conserved step in the activation of serine proteases, these results afford new perspective on the underlying thermodynamics of the transition from the zymogen to the active enzyme.

Thrombomodulin Binding Selects the Catalytically Active Form of Thrombin

“…Starting from a model of trypsin with the Nterminus artificially removed from the "Ile cleft", we were able to observe this insertion event in long aMD simulations. 6 As with thrombin, all crystallographically determined structures of trypsin have the N-terminus of the heavy chain buried (Protein Data Bank). It will be interesting to see whether solution analyses, perhaps by NMR, can reveal the dynamics of this critical region.…”

“…This salt bridge is thought to promote the formation of a correctly assembled S1 pocket of the active site and thereby full proteolytic activity. Using accelerated molecular dynamics (aMD) simulations, we recently showed that the N-terminus of trypsin inserts from a trypsinogen-like state into this pocket and remains in the pocket once inserted, indicating that the N-terminus is more stable inside the pocket than out . All crystal structures of α-thrombin show the N-terminus in this inserted conformation (Protein Data Bank).…”

Coevolved Positions Represent Key Functional Properties in the Trypsin-Like Serine Proteases Protein Family

“…More recently, the spontaneous insertion of Ile16 has been observed in an accelerated molecular dynamics, similarly to what was seen for PC1 and PC2 during our analysis. The Asp194 loop transition as seen for seen in PC 1 during the hybrid elastic network Brownian dynamics simulation was not observed in the accelerated molecular dynamics simulations . Given the very coarse level of detail of our simulations, further studies are needed in order to better understand these mechanisms.…”

Conformational Plasticity-Rigidity Axis of the Coagulation Factor VII Zymogen Elucidated by Atomistic Simulations of the N-Terminally Truncated Factor VIIa Protease Domain

“…In addition, three loop segments, activation loop 1, 2, and 3 (AL1–3), undergo stabilization [ 19 , 22 ]. The N-terminal insertion of trypsinogen activation has even been observed previously using accelerated molecular dynamics simulations [ 23 ]. While the activation transition of FVII is known to follow the same hallmark steps as trypsinogen-trypsin for it become catalytically active, many important details concerning FVII and the zymogen-like FVIIa are lacking.…”

“…It is unclear whether this regulatory framework is evolutionarily conserved among all species carrying FVIIa, something that has recently sparked some debate [ 69 , 70 , 71 ]. From the literature, we know that the energetic origins of the conformational changes induced by N-terminus insertion are both electrostatic and hydrophobic [ 23 , 72 , 73 ]. Thus, the zymogen-to-enzyme activation transition can be considered a cooperative process with distinct energetic and entropic contributions, making it difficult to delineate adequately on the basis of static structures.…”