The High Resolution Crystal Structure of a Native Thermostable Serpin Reveals the Complex Mechanism Underpinning the Stressed to Relaxed Transition

Kate F Fulton,Ashley M Buckle,Lisa D Cabrita,James A Irving,Rebecca E Butcher,Ian Smith,Shane Reeve,Arthur M Lesk,Stephen P Bottomley,Jamie Rossjohn,James C Whisstock

doi:10.1074/jbc.m410206200

Abstract

Serpins fold into a native metastable state and utilize a complex conformational change to inhibit target proteases. An undesirable result of this conformational flexibility is that most inhibitory serpins are heat sensitive, forming inactive polymers at elevated temperatures. However, the prokaryote serpin, thermopin, from Thermobifida fusca is able to function in a heated environment. We have determined the 1.8 A x-ray crystal structure of thermopin in the native, inhibitory conformation. A structural comparison with the previously determined 1.5 A structure of cleaved thermopin provides detailed insight into the complex mechanism of conformational change in serpins. Flexibility in the shutter region and electrostatic interactions at the top of the A beta-sheet (the breach) involving the C-terminal tail, a unique structural feature of thermopin, are postulated to be important for controlling inhibitory activity and triggering conformational change, respectively, in the native state. Here we have discussed the structural basis of how this serpin reconciles the thermodynamic instability necessary for function with the stability required to withstand elevated temperatures.

Highlights

IntroductionSerpins (peptidase inhibitor family I4) are the largest superfamily of protease inhibitors with over 800 members identified to date (1– 4)
Serpins are the largest superfamily of protease inhibitors with over 800 members identified to date (1– 4)
No electron density was observed in the region of the reactive center loop (RCL)

Summary

Introduction

Serpins (peptidase inhibitor family I4) are the largest superfamily of protease inhibitors with over 800 members identified to date (1– 4). Inhibitory serpins are unusual molecules that fold into a native metastable state and utilize a complex conformational change to achieve protease inhibition (4 – 6). Throughout the S to R transition, the protease remains covalently attached to the serpin via an acyl bond between the side chain of the active site serine (or cysteine) and the carbonyl oxygen of the P1 residue in the RCL. An undesirable consequence of this conformational flexibility is that serpins are susceptible to inappropriate conformational change, whereby the RCL of one molecule inserts into the A ␤-sheet of another, forming inactive long chain loop-sheet polymers (18, 19). Polymerization of native inhibitory serpins can be induced by mild heating (19, 25, 26)

Methods

Results

Conclusion