Amphiphilic Membrane Environments Regulate Enzymatic Behaviors of Salmonella Outer Membrane Protease

Siau Ling Kum; James C S Ho; Atul N Parikh; Bo Liedberg

doi:10.1021/acsbiomedchemau.1c00027

Amphiphilic Membrane Environments Regulate Enzymatic Behaviors of Salmonella Outer Membrane Protease

ACS Bio Med Chem Au. 2021 Dec 14;2(1):73-83. doi: 10.1021/acsbiomedchemau.1c00027. eCollection 2022 Feb 16.

Authors

Siau Ling Kum^{1

2}, James C S Ho^{1

2}, Atul N Parikh^{1

2

3

3}, Bo Liedberg^{1

2}

Affiliations

¹ Centre for Biomimetic Sensor Science, Nanyang Technological University, 50 Nanyang Drive, 637553 Singapore.
² School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Ave, 639798 Singapore.
³ Department of Chemistry and Department of Biomedical Engineering, University of California, Davis, California 95616, United States.

Abstract

The role of an amphiphilic environment in the functional regulation of integral membrane proteins is well appreciated but how specific amphiphilic surrounding influences the conformational plasticity and function of a protein is less obvious. We focus on the Salmonella phosphoglycerate transport system (pgt)-encoded outer membrane protease E (PgtE), which plays an important role in tissue infiltration and survival of Salmonella enterica. Despite our understanding of its physiological functions, elucidation of its enzymatic behavior in response to the immediate amphiphilic surrounding is lacking. We monitor the proteolytic activity of PgtE reconstituted in Zwittergent 3-12 detergent micelles or a 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) bilayer and examine factors that influence its activity. We find, to our surprise, that PgtE, which is thought to elicit a rapid response toward various substrates, showed hysteretic enzymatic behavior, characterized by a prominent lag phase prior to achieving the exponential steady state in its detergent-stabilized form as well as in the outer membrane embedded native state in live bacteria. The lag phase was abolished under three conditions: preformation of an inactive detergent-stabilized PgtE-substrate complex without lipopolysaccharide (LPS), LPS-bound detergent-stabilized PgtE that had reached steady state velocity, or PgtE reconstituted into a POPC bilayer environment. Interestingly, detergent- and bilayer-stabilized PgtE showed comparable steady-state activity. And strikingly, lipopolysaccharide (LPS) becomes nonessential for the activation of PgtE when the protein is reconstituted in the phospholipid bilayer, contrasting a long-standing notion that LPS is required for proteases belonging to the omptin family to be proteolytically active. These findings suggest intriguing biological nuances for the proteolytic function of PgtE that were not well appreciated previously and offer new perspectives that may generally be applicable for omptins.