Extreme parsimony in ATP consumption by 20S complexes in the global disassembly of single SNARE complexes

Changwon Kim,Reinhard Jahn,Gee Sung Eun,Min Ju Shon,Tae-Young Yoon,Sung Hyun Kim,Changbong Hyeon,Je-Kyung Ryu

doi:10.1038/s41467-021-23530-0

Abstract

Fueled by ATP hydrolysis in N-ethylmaleimide sensitive factor (NSF), the 20S complex disassembles rigid SNARE (soluble NSF attachment protein receptor) complexes in single unraveling step. This global disassembly distinguishes NSF from other molecular motors that make incremental and processive motions, but the molecular underpinnings of its remarkable energy efficiency remain largely unknown. Using multiple single-molecule methods, we found remarkable cooperativity in mechanical connection between NSF and the SNARE complex, which prevents dysfunctional 20S complexes that consume ATP without productive disassembly. We also constructed ATP hydrolysis cycle of the 20S complex, in which NSF largely shows randomness in ATP binding but switches to perfect ATP hydrolysis synchronization to induce global SNARE disassembly, minimizing ATP hydrolysis by non-20S complex-forming NSF molecules. These two mechanisms work in concert to concentrate ATP consumption into functional 20S complexes, suggesting evolutionary adaptations by the 20S complex to the energetically expensive mechanical task of SNARE complex disassembly.

Highlights

Membrane fusion is an energetically demanding process in eukaryotic cells[1,2], an evolutionary constraint that has forced membrane fusion proteins to deal with large scales of free energies[3,4,5]
By employing an array of single-molecule force and fluorescence spectroscopy techniques, we found that the 20S complex has developed two sophisticated layers of proofreading in its adenosine triphosphate (ATP) expenditure
With the improved temporal resolution of our magnetic tweezers, we observed that 20S complex-mediated disassembly exhibits a unique unfolding intermediate centered around the +4 layer (I20s) (Fig. 1i and Supplementary Fig. 2c), the intermediate not observed in the mechanical pulling of lone single sensitive factor attachment protein receptors (SNAREs) complexes

Summary

Introduction

Membrane fusion is an energetically demanding process in eukaryotic cells[1,2], an evolutionary constraint that has forced membrane fusion proteins to deal with large scales of free energies[3,4,5]. Soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) form a rigid four-helix bundle, called the SNARE complex, releasing huge free energy amounting to 65 kBT6–9. This explosive, single-step disassembly of SNARE complexes by NSF seems to have evolved to prevent reassembly of any partially unzipped SNARE complexes These observations position NSF as one of the first examples of an unfolding machine that induces a global disassembly of large protein complexes[28,29]. It distinguishes NSF from other molecular motors and unfoldases that typically induce linear or rotational translocation in incremental steps through many cycles of ATP binding and hydrolysis[30,31,32]. Our results collectively suggest the 20S complex has evolved sophisticated mechanisms for preventing unnecessary ATP expenditure while still accomplishing the energy-expensive mechanical task of SNARE complex disassembly

Methods

Results

Conclusion