Understanding Periperal Membrane Protein Interactions | BPS Thematic Meeting

Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Poster Abstracts

14-POS Board 14 STRUCTURAL STUDIES OF INTEGRAL MEMBRANE AND MEMBRANE ASSOCIATED ENZYMES IN THE AMINOARABINOSE BIOSYNTHETIC PATHWAY LINKED TO POLYMYXIN RESISTANCE IN GRAM-NEGATIVE BACTERIA Khuram U Ashraf 1,2 ; Ankita Punetha 1,2 ; Mariana Bunoro-Batista 3 ; Bertie Ansell 4 ; Phillip J Stansfeld 3 ; Vasileios I Petrou 1,2 ; 1 Rutgers New Jersey Medical School, Department of Microbiology, Biochemistry and Molecular Genetics, Newark, NJ, USA 2 Rutgers New Jersey Medical School, Center for Immunity and Inflammation, Newark, NJ, USA 3 University of Warwick, School of Life Sciences, Coventry, United Kingdom 4 University of Oxford, Department of Biochemistry, Oxford, United Kingdom Polymyxins are last-resort antibiotics that are used to treat multidrug resistant Gram-negative infections. But, enzymatic modification of the Lipid A moiety in the bacterial lipopolysaccharide decorating the outer membrane of Gram-negative bacteria leads to resistance to polymyxins and natural antimicrobial peptides (AMPs). In E. coli and Salmonella enterica, the most effective Lipid A modification for polymyxin resistance is the conjugation of aminoarabinose (L-Ara4N) at the 4’ phosphate of Lipid A. L-Ara4N is synthesized by a relay of cytoplasmic and membrane enzymes that function in sequence, known as the aminoarabinose biosynthetic pathway. The polyprenol phosphate glycosyltransferase ArnC is the first membrane enzyme to act in the pathway and is responsible for loading a formylated form of L-Ara4N to the carrier lipid undecaprenyl phosphate (UndP). Thus, it represents a transition point in the pathway, enabling the association of the aminoarabinose sugar with bacterial inner membranes. Subsequently, the product of ArnC is deformylated by the membrane-associated enzyme ArnD, which prevents backward reaction, rendering the activity of the glycosyltransferase ArnC effectively irreversible. We have used single-particle cryo-electron microscopy (cryo-EM) to characterize the structure of nanodisc-embedded ArnC, in an apo and partial donor substrate-bound state, and perform preliminary characterization of the membrane-associated enzyme ArnD in lipidic nanodiscs. The initial cryo-EM analysis reveals a dimeric state for nanodisc-embedded ArnD. We further utilize microscale thermophoresis (MST) to characterize interactions of ArnC and ArnD with catalytic metal ions, providing insights on the molecular determinants of their function. We use coarse grained (CG) simulations and the LipIDens pipeline to characterize interactions of ArnC with lipids based on lipid-like densities observed in the cryo-EM map. Finally, we perform atomistic simulations of ArnC with both of its substrates to provide insights on interactions of the lipid acceptor UndP within the cytoplasmic glycosyltransferase domain and the catalytic mechanism employed by ArnC.

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